Hybrid tomato plant HMX4909

ABSTRACT

A novel hybrid tomato plant, designated HMX4909 is disclosed. The invention relates to the seeds of tomato hybrid HMX4909, to the plants and plant parts of hybrid tomato HMX4909, and to methods for producing a tomato plant by crossing the hybrid tomato HMX4909 with itself or another tomato plant. The invention further relates to methods for producing a tomato plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other tomato plants derived from the hybrid tomato plant HMX4909.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to, and the benefit of U.S.Provisional Patent Application No. 62/118,918, filed Feb. 20 2015, whichis herein incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture, to new anddistinctive hybrid tomato plants, such as hybrid plants designatedHMX4909 and to methods of making and using such hybrids.

BACKGROUND OF THE INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Tomato is an important and valuable vegetable crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding tomatohybrids that are agronomically sound. The reasons for this goal are tomaximize the amount of fruits produced on the land used (yield) as wellas to improve the plant and fruit agronomic qualities. To accomplishthis goal, the tomato breeder must select and develop tomato plants thathave the traits that result in superior parental lines that combine toproduce superior hybrids.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope.

In various embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

According to the invention, there are provided novel hybrid tomatoes,designated HMX4909.

This invention thus relates to the seeds of hybrid tomato designatedHMX4909, to the plants or parts thereof of hybrid tomato designatedHMX4909, to plants or parts thereof consisting essentially of thephenotypic and morphological characteristics of hybrid tomato designatedHMX4909, and/or having all the physiological and morphologicalcharacteristics of hybrid tomato designated HMX4909, and/or having oneor more or all of the characteristics of hybrid tomato designatedHMX4909 listed in Table 1 including but not limited to as determined atthe 5% significance level when grown in the same environmentalcondition, and/or having the physiological and morphologicalcharacteristics of hybrid tomato designated HMX4909 listed in Table 1including but not limited to as determined at the 5% significance levelwhen grown in the same environmental condition and/or having all thephysiological and morphological characteristics of hybrid tomatodesignated HMX4909 listed in Table 1 when grown in the sameenvironmental condition. The invention also relates to variants, mutantsand trivial modifications of the seed or plant of hybrid tomatodesignated HMX4909.

Plant parts of the hybrid tomato plant of the present invention are alsoprovided, such as, a scion, a rootstock, a fruit, leaf, flower, cell,pollen or ovule obtained from the hybrid plant. The present inventionprovides fruits of the hybrid tomato of the present invention. Suchfruits could be used as fresh products for consumption or in processesresulting in processed products such as juice, prepared fruit cuts,canned tomatoes, pastes, sauces, puree, catsups and the like. All suchproducts are part of the present invention.

The plants and seeds of the present invention include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act of The United States of America, e.g., avariety that is predominantly derived from hybrid tomato designatedHMX4909 or from a variety that i) is predominantly derived from hybridtomato designated HMX4909, while retaining the expression of theessential characteristics that result from the genotype or combinationof genotypes of hybrid tomato designated HMX4909; ii) is clearlydistinguishable from hybrid tomato designated HMX4909; and iii) exceptfor differences that result from the act of derivation, conforms to theinitial variety in the expression of the essential characteristics thatresult from the genotype or combination of genotypes of the initialvariety.

In another aspect, the present invention provides regenerable cells. Insome embodiments, the regenerable cells are for use in tissue culture ofhybrid tomato designated HMX4909. In some embodiments, the tissueculture is capable of regenerating plants consisting essentially of thephenotypic and morphological characteristics of hybrid tomato designatedHMX4909, and/or having all the phenotypic and morphologicalcharacteristics of hybrid tomato designated HMX4909, and/or having thephysiological and morphological characteristics of hybrid tomatodesignated HMX4909, and/or having the characteristics of hybrid tomatodesignated HMX4909. In some embodiments, the plant parts and cells usedto produce such tissue cultures will be embryos, meristematic cells,seeds, callus, pollen, leaves, anthers, pistils, roots, root tips,stems, petioles, fruits, cotyledons, hypocotyls, ovaries, seed coat,fruits, endosperm, flowers, axillary buds or the like. Protoplastsproduced from such tissue culture are also included in the presentinvention. The tomato shoots, roots and whole plants regenerated fromthe tissue culture, as well as the fruits produced by said regeneratedplants are also part of the invention. In some embodiments, the wholeplants regenerated from the tissue culture have one, more than one, orall of the physiological and morphological characteristics of tomatohybrid designated HMX4909 listed in Table 1 including but not limited towhen grown in the same environmental condition.

The invention also discloses methods for vegetatively propagating aplant of the present invention. In some embodiments, the methodscomprise collecting a part of a hybrid tomato designated HMX4909 andregenerating a plant from said part. In some embodiments, the part canbe for example a stem cutting that is rooted into an appropriate mediumaccording to techniques known by the one skilled in the art. Plants,part parts and fruits thereof produced by such methods are also includedin the present invention. In another aspect, the plants and fruitsthereof produced by such methods consist essentially of the phenotypicand morphological characteristics of hybrid tomato designated HMX4909,and/or having all the phenotypic and morphological characteristics ofhybrid tomato designated HMX4909 and/or having the physiological andmorphological characteristics of hybrid tomato designated HMX4909 and/orhaving the characteristics of hybrid tomato designated HMX4909. In someembodiments, plants produced by such methods consist of one, more thanone, or all phenotypic and morphological characteristics of tomatohybrid designated listed in Table 1 including but not limited to whengrown in the same environmental condition.

Further included in the invention are methods for producing fruits fromthe hybrid tomato designated HMX4909. In some embodiments, the methodscomprise growing a hybrid tomato designated HMX4909 to produce a tomatofruit. In some embodiments, the methods further comprise harvesting thehybrid tomato fruit. Such fruits are part of the present invention.

Also included in this invention are methods for producing a tomatoplant. In some embodiments, the tomato plant is produced by crossing thehybrid tomato designated HMX4909 with itself or another tomato plant. Insome embodiments, the other plant can be a tomato hybrid or line. Whencrossed with an inbred line, in some embodiments, a “three-way cross” isproduced. When crossed with itself or with another, different hybridtomato, in some embodiment, a “four-way” cross is produced. Such threeand four-way hybrid seeds and plants produced by growing said three andfour-way hybrid seeds are included in the present invention. Methods forproducing a three and four-way hybrid tomato seed comprising crossinghybrid tomato designated HMX4909 tomato plant with a different tomatoline or hybrid and harvesting the resultant hybrid tomato seed are alsopart of the invention. The hybrid tomato seeds produced by the methodcomprising crossing hybrid tomato designated HMX4909 tomato plant with adifferent tomato plant and harvesting the resultant hybrid tomato seedare included in the invention, as are included the hybrid tomato plantor parts thereof and seeds produced by said grown hybrid tomato plants.

Further included in the invention are methods for producing tomato seedsand plants made thereof. In some embodiments, the methods compriseself-pollinating the hybrid tomato designated HMX4909 and harvesting theresultant hybrid seeds. Tomato seeds produced by such method are alsopart of the invention.

In another embodiment, this invention relates to methods for producing ahybrid tomato designated HMX4909 from a collection of seeds. In someembodiments, the collection contains one or both inbred line of hybridtomato designated HMX4909 seeds and hybrid seeds of HMX4909 Such acollection of seeds might be a commercial bag of seeds. In someembodiments, said methods comprise planting the collection of seeds.When planted, the collection of seeds will produce inbred parent linesof hybrid tomato HMX4909 and hybrid plants from the hybrid seeds ofHMX4909. In some embodiments, said inbred parent lines of hybrid tomatodesignated HMX4909 plants are identified as having a decreased vigorcompared to the other plants grown from the collection of seeds. In someembodiments, said decreased vigor is due to the inbreeding depressioneffect and can be identified for example by a less vigorous appearancefor vegetative and/or reproductive characteristics including a slightreduction in plant size, slightly smaller fruit size, possibly latermaturity and/or a possible reduction in yield. In some embodiments,seeds of the inbred lines of the hybrid tomato HMX4909 are collected, ifnew inbred plants thereof are grown and crossed in a controlled mannerwith each other, the hybrid tomato HMX4909 will be recreated.

This invention also relates to methods for producing other tomato plantsderived from hybrid tomato HMX4909 and to the tomato plants derived bythe use of those methods.

In some embodiments, such methods for producing a tomato plant derivedfrom the hybrid variety HMX4909 comprise (a) self-pollinating the hybridtomato HMX4909 plant at least once to produce a progeny plant derivedfrom tomato hybrid HMX4909; In some embodiments, the methods furthercomprise (b) crossing the progeny plant derived from tomato hybridHMX4909 with itself or a second tomato plant to produce a seed of aprogeny plant of a subsequent generation; In some embodiments, themethods further comprise (c) growing the progeny plant of the subsequentgeneration; In some embodiments, the methods further comprise (d)crossing the progeny plant of the subsequent generation with itself or asecond tomato plant to produce a tomato plant further derived from thehybrid tomato HMX4909. In further embodiments, steps (b), (c) and/or (d)are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, or more generation toproduce a tomato plant derived from the hybrid tomato variety HMX4909.In some embodiments, within each crossing cycle, the second plant is thesame plant as the second plant in the last crossing cycle. In someembodiments, within each crossing cycle, the second plant is differentfrom the second plant in the last crossing cycle.

Another method for producing a tomato plant derived from the hybridvariety HMX4909, comprises the steps of: (a) crossing the hybrid tomatoHMX4909 plant with a second tomato plant to produce a progeny plantderived from tomato hybrid HMX4909; In some embodiments, the methodsfurther comprise (b) crossing the progeny plant derived from tomatohybrid HMX4909 with itself or a second tomato plant to produce a seed ofa progeny plant of a subsequent generation; In some embodiments, themethods further comprise (c) growing the progeny plant of the subsequentgeneration; In some embodiments, the methods further comprise (d)crossing the progeny plant of the subsequent generation with itself or asecond tomato plant to produce a tomato plant derived from the hybridtomato variety HMX4909. In a further embodiment, steps (b), (c) and/or(d) are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, or more generationto produce a tomato plant derived from the hybrid tomato varietyHMX4909. In some embodiments, within each crossing cycle, the secondplant is the same plant as the second plant in the last crossing cycle.In some embodiments, within each crossing cycle, the second plant isdifferent from the second plant in the last crossing cycle.

More specifically, the invention comprises methods for producing a malesterile tomato plant, an herbicide resistant tomato plant, an insectresistant tomato plant, a disease resistant tomato plant, awater-stress-tolerant plant, a heat stress tolerant plants, an improvedshelf life tomato plant, a tomato plant with increased sweetness andflavor, a tomato plant with increased sugar content, a tomato plant withdelayed senescence or controlled ripening and/or plants with improvedsalt tolerance. In some embodiments, said methods comprise transformingthe hybrid designated HMX4909 tomato plant with nucleic acid moleculesthat confer male sterility, herbicide resistance, insect resistance,disease resistance, water-stress tolerance, heat stress tolerance,increased shelf life, increased sweetness and flavor, increased sugarcontent, delayed senescence or controlled ripening and/or improved salttolerance, respectively. The transformed tomato plants or parts thereof,obtained from the provided methods, including for example a male steriletomato plant, an herbicide resistant tomato plant, an insect resistanttomato plant, a disease resistant tomato plant, a tomato with waterstress tolerance, a tomato with heat stress tolerance, a tomato plantswith increased sweetness and flavor, a tomato plants with increasedsugar content, a tomato plants with delayed senescence or controlledripening or a tomato plants with improved salt tolerance are included inthe present invention. Plants may display one or more of the abovelisted traits. For the present invention and the skilled artisan,disease is understood to include, but not limited to fungal diseases,viral diseases, bacterial diseases, mycoplasm diseases, or other plantpathogenic diseases and a disease resistant plant will encompass a plantresistant to fungal, viral, bacterial, mycoplasm, and other plantpathogens.

In another aspect, the present invention provides methods of introducingone or more desired trait(s) into the hybrid tomato HMX4909 and plantsor seeds obtained from such methods. The desired trait(s) may be, butnot exclusively, a single gene. In some embodiments, the gene is adominant allele. In some embodiments, the gene is a partially dominantallele. In some embodiments, the gene is a recessive allele. In someembodiments, the transferred gene or genes will confer such traits asmale sterility, herbicide resistance, insect resistance, resistance forbacterial, fungal, mycoplasma or viral disease, improved shelf life,water-stress tolerance, delayed senescence or controlled ripening,enhanced nutritional quality such as increased sugar content orincreased sweetness, enhanced plant quality such as improved drought orsalt tolerance, enhanced plant vigor or improve fresh cut application,specific aromatic compounds, specific volatiles, flesh texture; specificnutritional components and/or long shelf life (LSL). The gene or genesmay be naturally occurring or tomato gene(s), mutant(s) or transgene(s)introduced through genetic engineering techniques. In some embodiments,the methods for introducing the desired trait(s) comprise backcrossingprocess making use of a series of backcrosses to at least one of theparent lines of hybrid tomato HMX4909 during which the desired trait(s)is maintained by selection. The single gene conversion plants that canbe obtained by the methods are included in the present invention.

When using a transgene, in some embodiments, the trait is generally notincorporated into each newly developed hybrid such as HMX4909 by directtransformation. Rather, the more typical method used by breeders ofordinary skill in the art to incorporate the transgene is to take a linealready carrying the transgene and to use such line as a donor line totransfer the transgene into one or more of the parents of the newlydeveloped hybrid. The same would apply for a naturally occurring traitor one arising from spontaneous or induced mutations. In someembodiments, the backcross breeding process comprises (a) crossing oneof the parental inbred line plants of HMX4909 with plants of anotherline that comprise the desired trait(s) to produce F1 progeny plants; Insome embodiments, the process further comprises (b) selecting the F1progeny plants that have the desired trait(s); In some embodiments, theprocess further comprises (c) crossing the selected F1 progeny plantswith the parental inbred tomato lines of hybrid HMX4909 plants toproduce backcross progeny plants; In some embodiments, the processfurther comprises (d) selecting for backcross progeny plants that havethe desired trait(s) and physiological and morphological characteristicsof the tomato parental inbred line of hybrid tomato HMX4909 to produceselected backcross progeny plants; In some embodiments, the processfurther comprises (e) repeating steps (c) and (d) one, two, three, four,five six, seven, eight, nine or more times in succession to produceselected, second, third, fourth, fifth, sixth, seventh, eighth, ninth orhigher backcross progeny plants that have the desired trait(s) andconsist essentially of the phenotypic and morphological characteristicsof the parental inbred tomato line of tomato HMX4909, and/or have allthe phenotypic and morphological characteristics of the parental tomatoinbred line of hybrid tomato HMX4909, and/or the physiological andmorphological characteristics of the parental inbred tomato line oftomato hybrid HMX4909 as determined in Table 1, including but notlimited to when grown in the same environmental condition or includingbut not limited to at a 5% significance level when grown in the sameenvironmental conditions. The tomato plants or seed produced by themethods are also part of the invention, as are the hybrid tomato HMX4909plants that comprised the desired trait. Backcrossing breeding methods,well known to one skilled in the art of plant breeding will be furtherdeveloped in subsequent parts of the specification.

In an embodiment of this invention is a method of making a backcrossconversion of hybrid tomato HMX4909. In some embodiments, the methodcomprises crossing one of the parental tomato inbred line plants ofhybrid HMX4909 with a donor plant comprising a mutant gene(s), anaturally occurring gene(s), or transgene(s) conferring one or moredesired trait to produce F1 progeny plants; In some embodiments, themethod further comprises selecting the F1 progeny plant comprising thenaturally occurring gene(s), mutant gene(s) or transgene(s) conferringthe one or more desired trait; In some embodiments, the method furthercomprises backcrossing the selected progeny plant to the parental tomatoinbred line plants of hybrid HMX4909. This method may further comprisethe step of obtaining a molecular marker profile of the parental tomatoinbred line plants of hybrid HMX4909 and using the molecular markerprofile to select for the progeny plant with the desired trait and themolecular marker profile of the parental tomato inbred line plants ofhybrid HMX4909. In some embodiments, this method further comprisescrossing the backcross progeny plant HMX4909 containing the naturallyoccurring gene(s), the mutant gene(s) or the transgene(s) conferring theone or more desired trait with the second parental inbred tomato lineplants of hybrid tomato HMX4909 in order to produce the hybrid tomatoHMX4909 comprising the naturally occurring gene(s), the mutant gene(s)or transgene(s) conferring the one or more desired trait. The plants orparts thereof produced by such methods are also part of the presentinvention.

In some embodiments of the invention, the number of loci that may bebackcrossed into the parental tomato inbred line of hybrid HMX4909 is atleast 1, 2, 3, 4, 5, or more. A single locus may contain severaltransgenes, such as a transgene for disease resistance that, in the sameexpression vector, also contains a transgene for herbicide resistance.The gene for herbicide resistance may be used as a selectable markerand/or as a phenotypic trait. A single locus conversion of site specificintegration system allows for the integration of multiple genes at theconverted locus. A single locus conversion also allows for making one ormore site specific changes to the plant genome. In some embodiments, thesingle locus conversion is performed by genome editing, a.k.a. genomeediting with engineered nucleases (GEEN). In some embodiments, thegenome editing comprises using one or more engineered nucleases. In someembodiments, the engineered nucleases include, but are not limited toZinc finger nucleases (ZFNs), Transcription Activator-Like EffectorNucleases (TALENs), the CRISPR/Cas system, and engineered meganucleasere-engineered homing endonucleases. In some embodiments, the singlelocus conversion changes one or several nucleic acids of the plantgenome.

The invention further provides methods for developing tomato plants in atomato plant breeding program using plant breeding techniques includingbut not limited to, recurrent selection, backcrossing, pedigreebreeding, molecular marker (Isozyme Electrophoresis, RestrictionFragment Length Polymorphisms (RFLPs), Randomly Amplified PolymorphicDNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), andSimple Sequence Repeats (SSRs) which are also referred to asMicrosatellites, Single Nucleotide Polymorphism (SNP) enhancedselection, genetic marker enhanced selection and transformation. Seeds,tomato plants, and parts thereof produced by such breeding methods arealso part of the invention.

The invention also relates to variants, mutants and trivialmodifications of the seed or plant of the tomato hybrid HMX4909 orinbred parental lines thereof. Variants, mutants and trivialmodifications of the seed or plant of hybrid tomato HMX4909 or inbredparental lines thereof can be generated by methods available to oneskilled in the art, including but not limited to, mutagenesis (e.g.,chemical mutagenesis, radiation mutagenesis, transposon mutagenesis,insertional mutagenesis, signature tagged mutagenesis, site-directedmutagenesis, and natural mutagenesis), knock-outs/knock-ins, antisenseand RNA interference. For more information of mutagenesis in plants,such as agents, protocols, see Acquaah et al. (Principles of plantgenetics and breeding, Wiley-Blackwell, 2007, ISBN 1405136464,9781405136464, which is herein incorporated by reference in its entity).

The invention also relates to a mutagenized population of the hybridtomato HMX4909 and methods of using such populations. In someembodiments, the mutagenized population can be used in screening for newtomato plants which comprise one or more or all of the morphological andphysiological characteristics of hybrid tomato HMX4909. In someembodiments, the new tomato plants obtained from the screening processcomprise all of the morphological and physiological characteristics ofthe tomato hybrid HMX4909 and one or more additional or differentmorphological and physiological characteristics that the tomato hybridHMX4909 does not have.

This invention also is directed to methods for producing a tomato plantby crossing a first parent tomato plant with a second parent tomatoplant wherein either the first or second parent tomato plant is a hybridtomato plant of HMX4909. Further, both first and second parent tomatoplants can come from the hybrid tomato plant HMX4909. Further, thehybrid tomato plant HMX4909 can be self-pollinated i.e. the pollen of ahybrid tomato plant HMX4909 can pollinate the ovule of the same hybridtomato plant HMX4909. When crossed with another tomato plant, a hybridseed is produced. Such methods of hybridization and self-pollination arewell known to those skilled in the art of breeding.

An inbred tomato line such as one of the parental lines of hybrid tomatoHMX4909 has been produced through several cycles of self-pollination andis therefore to be considered as a homozygous line. An inbred line canalso be produced though the dihaploid system which involves doubling thechromosomes from a haploid plant or embryo thus resulting in an inbredline that is genetically stable (homozygous) and can be reproducedwithout altering the inbred line: Haploid plants could be obtained fromhaploid embryos that might be produced from microspores, pollen, anthercultures or ovary cultures or spontaneous haploidy. The haploid embryosmay then be doubled by chemical treatments such as by colchicine or bedoubled autonomously. The haploid embryos may also be grown into haploidplants and treated to induce the chromosome doubling. In either case,fertile homozygous plants are obtained. A hybrid variety is classicallycreated through the fertilization of an ovule from an inbred parentalline by the pollen of another, different inbred parental line. Due tothe homozygous state of the inbred line, the produced gametes carry acopy of each parental chromosome. As both the ovule and the pollen bringa copy of the arrangement and organization of the genes present in theparental lines, the genome of each parental line is present in theresulting F1 hybrid, theoretically in the arrangement and organizationcreated by the plant breeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross shall be stable. The F1 hybrid is then acombination of phenotypic characteristics issued from two arrangementand organization of genes, both created by a man skilled in the artthrough the breeding process.

Still further, this invention also is directed to methods for producinga tomato plant derived from hybrid tomato HMX4909 by crossing hybridtomato plant HMX4909 with a second tomato plant. In some embodiments,the methods further comprise obtaining a progeny seed from the cross. Insome embodiments, the methods further comprise growing the progeny seed,and possibly repeating the crossing and growing steps with the tomatohybrid plant HMX4909-derived plant from 0 to 7 or more times. Thus, anysuch methods using the hybrid tomato plant HMX4909 are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using hybrid tomato plantHMX4909 as a parent are within the scope of this invention, includingplants derived from hybrid tomato plant HMX4909. In some embodiments,such plants have one or more or all physiological and morphologicalcharacteristics of the tomato hybrid plant HMX4909. In some embodimentssuch plants might exhibit additional and desired characteristics ortraits such as high seed yield, high seed germination, seedling vigor,early fruit maturity, high fruit yield, ease of fruit setting, diseasetolerance or resistance, and adaptability for soil and climateconditions. Consumer-driven traits, such as a preference for a givenfruit size, shape, color, texture, taste, fruit firmness, brix and sugarcontent are other traits that may be incorporated into new tomato plantsdeveloped by this invention.

A tomato plant can also be propagated vegetatively. A part of the plant,for example a shoot tissue, is collected, and a new plant is obtainedfrom the part. Such part typically comprises an apical meristem of theplant. The collected part is transferred to a medium allowingdevelopment of a plantlet, including for example rooting or developmentof shoots, or is grafted onto a tomato plant or a rootstock prepared tosupport growth of shoot tissue. This is achieved using methods wellknown in the art. Accordingly, in one embodiment, a method ofvegetatively propagating a plant of the present invention comprisescollecting a part of a plant according to the present invention, e.g. ashoot tissue, and obtaining a plantlet from said part. In oneembodiment, a method of vegetatively propagating a plant of the presentinvention comprises: a) collecting tissue of a plant of the presentinvention; b) rooting said proliferated shoots to obtain rootedplantlets. In one embodiment, a method of vegetatively propagating aplant of the present invention comprises: a) collecting tissue of aplant of the present invention; b) cultivating said tissue to obtainproliferated shoots; c) rooting said proliferated shoots to obtainrooted plantlets. In one embodiment, such method further comprisesgrowing a plant from said plantlets. In one embodiment, a fruit isharvested from said plant. In one embodiment, the fruit is processedinto products such as canned tomato, juice, fresh or prepared cutfruits, pastes, sauces, puree, catsups and the like.

In some embodiments, the present invention teaches a seed of hybridtomato designated HMX4909, wherein a representative sample of seed ofsaid hybrid is deposited under NCIMB No. 42543.

In some embodiments, the present invention teaches a tomato plant, or apart thereof, produced by growing the deposited HMX4909 seed.

In some embodiments, the present invention teaches tomato plant parts,wherein the tomato part is selected from the group consisting of: aleaf, a flower, a fruit, an ovule, pollen, a cell, a rootstock, and ascion.

In some embodiments, the present invention teaches a tomato plant, or apart thereof, having all of the characteristics of hybrid HMX4909 aslisted in Table 1 of this application including but not limited to whengrown in the same environmental condition.

In some embodiments, the present invention teaches a tomato plant, or apart thereof, having all of the physiological and morphologicalcharacteristics of hybrid HMX4909, wherein a representative sample ofseed of said hybrid was deposited under NCIMB No. 42543.

In some embodiments, the present invention teaches a tissue culture ofregenerable cells produced from the plant or plant part grown from thedeposited HMX4909 seed, wherein cells of the tissue culture are producedfrom a plant part selected from the group consisting of protoplasts,embryos, meristematic cells, callus, pollen, ovules, flowers, seeds,leaves, roots, root tips, anthers, stems, petioles, fruits, axillarybuds, cotyledons and hypocotyls. In some embodiments, the plant partincludes protoplasts produced from a plant grown from the depositedHMX4909 seed.

In some embodiments, the present invention teaches a tomato plantregenerated from the tissue culture from a plant grown from thedeposited HMX4909 seed, said plant having the characteristics of hybridHMX4909, wherein a representative sample of seed of said hybrid isdeposited under NCIMB No. 42543.

In some embodiments, the present invention teaches a tomato fruitproduced from the plant grown from the deposited HMX4909 seed.

In some embodiments, methods of producing said tomato fruit comprise a)growing the tomato plant from deposited HMX4909 seed to produce a tomatofruit, and b) harvesting said tomato fruit. In some embodiments, thepresent invention also teaches a tomato fruit produced by the method ofproducing tomato fruit as described above.

In some embodiments, the present invention teaches methods for producinga tomato seed comprising crossing a first parent tomato plant with asecond parent tomato plant and harvesting the resultant tomato seed,wherein said first parent tomato plant and/or second parent tomato plantis the tomato plant produced from the deposited HMX4909 seed or a tomatoplant having all of the characteristics of tomato hybrid HMX4909 aslisted in Table 1 including but not limited to when grown in the sameenvironmental condition.

In some embodiments, the present invention teaches methods for producinga tomato seed comprising self-pollinating the tomato plant grown fromthe deposited HMX4909 seed and harvesting the resultant tomato seed.

In some embodiments, the present invention teaches the seed produced byany of the above described methods.

In some embodiments, the present invention teaches methods ofvegetatively propagating the tomato plant grown from the depositedHMX4909 seed, said method comprising a) collecting part of a plant grownfrom the deposited HMX4909 seed and b) regenerating a plant from saidpart.

In some embodiments, the method further comprises harvesting a fruitfrom said vegetatively propagated plant.

In some embodiments, the present invention teaches the plant and thefruit of plants vegetatively propagated from plant parts of plants grownfrom the deposited HMX4909 seed.

In some embodiments, the present invention teaches methods of producinga tomato plant derived from the hybrid variety HMX4909, the methodscomprise (a) self-pollinating the plant of grown from the depositedHMX4909 seed at least once to produce a progeny plant derived fromtomato hybrid HMX4909; In some embodiments, the method further comprises(b) crossing the progeny plant derived from tomato hybrid HMX4909 withitself or a second tomato plant to produce a seed of a progeny plant ofa subsequent generation, and; (c) growing the progeny plant of thesubsequent generation from the seed, and crossing the progeny plant of asubsequent generation with itself or a second tomato plant to produce atomato plant derived from the hybrid tomato variety HMX4909. In someembodiments said methods further comprise the step of: (d) repeatingsteps b) or c) for at least 1, 2, 3, 4, 5, 6, 7, or more generation toproduce a tomato plant derived from the hybrid tomato variety HMX4909.

In some embodiments, the present invention teaches methods of producinga tomato plant derived from the hybrid variety HMX4909, the methodscomprising (a) crossing the plant grown from the deposited HMX4909 seedwith a second tomato plant to produce a progeny plant derived fromtomato hybrid HMX4909. In some embodiments, the method furthercomprises: (b) crossing the progeny plant derived from tomato hybridHMX4909 with itself or a second tomato plant to produce a seed of aprogeny plant of a subsequent generation, and (c) growing the progenyplant of the subsequent generation from the seed; (d) crossing theprogeny plant of the subsequent generation with itself or a secondtomato plant to produce a tomato plant derived from the hybrid tomatovariety HMX4909. In some embodiments said methods further comprise thesteps of: (e) repeating step (b), (c) and/or (d) for at least 1, 2, 3,4, 5, 6, 7 or more generation to produce a tomato plant derived from thehybrid tomato variety HMX4909.

In some embodiments, the present invention teaches methods for producinga transgenic tomato plant, the methods comprising crossing a firsttomato plant grown from the deposited HMX4909 seed with a second tomatoplant containing a transgene, wherein the transgene of said secondtomato plant is integrated into the genome of the tomato plant progenyresulting from said cross, and wherein the transgene confers said tomatoplant progeny with at least one trait selected from the group consistingof male sterility, male fertility, herbicide resistance, insectresistance, disease resistance, increased sweetness, increased sugarcontent, increased flavor, improved ripening control, and improved salttolerance when compared with plants not comprising such transgene. Insome embodiment, the plant not comprising such transgene is tomatohybrid HMX4909.

In some embodiments, the present invention teaches methods for producinga transgenic tomato plant, the methods comprising transforming at leastone transgene into a hybrid tomato HMX4909 plant, or a plant part or aplant cell thereof or parental line used for producing the hybrid tomatoplant HMX4909, a sample seed of said hybrid having been deposited underNCIMB Accession No. 42543, thereby producing a transgenic tomato plant.

In some embodiments, the present invention teaches tomato plants andtomato fruits produced by any of the above-described methods ofproducing transgenic tomato. Thus, in some embodiments, the presentinvention teaches a plant grown from the deposited HMX4909 seed, furthercomprising a transgene. In some embodiments, said transgenic tomatoplants comprise transgenes, which confer said plant with a traitselected from the group consisting of male sterility, male fertility,herbicide resistance, insect resistance, disease resistance, increasedsweetness, and increased sugar content, increased flavor, improvedripening control, and improved salt tolerance when compared to plantsnot comprising such transgene. In some embodiments, the plant notcomprising such transgene is a tomato hybrid HMX4909.

In some embodiments, the present invention teaches plants grown from thedeposited HMX4909 seed wherein said plants comprise at least one singlelocus conversion. In some embodiments said single locus conversionconfers said plants with a trait selected from the group consisting ofmale sterility, male fertility, herbicide resistance, insect resistance,disease resistance, increased sweetness, increased sugar content,increased flavor, improved ripening control, long shelf life, specificaromatic compounds and improved salt tolerance when compared to plantsnot comprising such single locus conversion. In some embodiments, theplant not comprising such locus conversion is tomato hybrid HMX4909. Insome embodiments, the at least one single locus conversion is anartificially mutated gene.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Collection of seeds. In the context of the present invention acollection of seeds is a grouping of seeds mainly containing similarkind of seeds, for example hybrid seeds having the inbred line of theinvention as a parental line, but that may also contain, mixed togetherwith this first kind of seeds, a second, different kind of seeds, of oneof the inbred parent lines, for example the inbred line of the presentinvention. A commercial bag of hybrid seeds having the inbred line ofthe invention as a parental line and containing also the inbred lineseeds of the invention would be, for example such a collection of seeds.

Determinate tomatoes. Determinate tomatoes are tomato varieties thatcome to fruit all at once, then stop bearing. They are best suited forcommercial growing and mechanical harvesting since they can be harvestedall at once.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

Flesh color: In the context of the present invention, the flesh color isthe color of the tomato flesh that can range from orange-red to dark redwhen at ripe stage (harvest maturity).

Field holding ability: Field holding ability is the ability for fruitquality to maintain even after fruit is ripe (has turned red).

Grafting. Grafting is the operation by which a rootstock is grafted witha scion. The primary motive for grafting is to avoid damages bysoil-born pest and pathogens when genetic or chemical approaches fordisease management are not available. Grafting a susceptible scion ontoa resistant rootstock can provide a resistant cultivar without the needto breed the resistance into the cultivar. In addition, grafting mayenhance tolerance to abiotic stress, increase yield and result in moreefficient water and nutrient uses.

Immunity to disease(s) and or insect(s). A tomato plant which is notsubject to attack or infection by specific disease(s) and or insect(s)is considered immune.

Intermediate/Moderate resistance to disease(s) and or insect(s). Atomato plant that restricts the growth and development of specificdisease(s) and or insect(s), but may exhibit a greater range of symptomsor damage compared to high/standard resistant plants. Intermediateresistant plants will usually show less severe symptoms or damage thansusceptible plant varieties when grown under similar environmentalconditions and/or specific disease(s) and or insect(s) pressure, but mayhave heavy damage under heavy pressure. Intermediate resistant tomatoplants are not immune to the disease(s) and or insect(s).

Maturity. Maturity is the number of days from transplanting toharvesting.

Plant adaptability. A plant having good plant adaptability means a plantthat will perform well in different growing conditions and seasons.

Plant Cell. Plant cell, as used herein includes plant cells whetherisolated, in tissue culture or incorporated in a plant or plant part.

Plant Part. As used herein, the term plant includes plant cells, plantprotoplasts, plant cell tissue cultures from which tomato plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,seeds, fruit, rootstock, scions, stems, roots, anthers, pistils, roottips, leaves, meristematic cells, axillary buds, hypocotyls cotyledons,ovaries, seed coat endosperm and the like.

Predicted paste bostwick. The predicted paste bostwick is the calculatednumber with the brix and Bostwick reading using the following formula:Predicted paste bostwick=−11.53+(1.64*juice brix)+(0.5*juice bostwick).

Quantitative Trait Loci (QTL) Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Resistance to disease(s) and or insect(s). A tomato plant that restrictshighly the growth and development of specific disease(s) and orinsect(s) under normal disease(s) and or insect(s) attack pressure whencompared to susceptible plants. These tomato plants can exhibit somesymptoms or damage under heavy disease(s) and or insect(s) pressure.Resistant tomato plants are not immune to the disease(s) and orinsect(s).

Rootstock. A rootstock is the lower part of a plant capable of receivinga scion in a grafting process.

Scion. A scion is the higher part of a plant capable of being graftedonto a rootstock in a grafting process.

Semi-erect habit. A semi-erect plant has a combination of lateral andupright branching and has an intermediate type habit between a prostateplant habit, having laterally growing branching with fruits most of thetime on the ground and an erect plant habit with branching goingstraight up with fruit being off the ground.

Single gene converted (conversion). Single gene converted (conversion)plants refer to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a plant are recovered in additionto the single gene transferred into the plant via the backcrossingtechnique or via genetic engineering.

Soluble Solids. Soluble solids refers to the percent of solid materialthat dissolve into tomato puree or juice, the vast majority of which issugars. Soluble solids are directly related to finished processedproduct yield of paste and sauce. Soluble solids are estimated with arefractometer, and measured as degrees brix.

Susceptible to disease(s) and or insect(s). A tomato plant that issusceptible to disease(s) and or insect(s) is defined as a tomato plantthat has the inability to restrict the growth and development ofspecific disease(s) and or insect(s). Plants that are susceptible willshow damage when infected and are more likely to have heavy damage undermoderate levels of specific disease(s) and or insect(s).

Tolerance to abiotic stresses. A tomato plant that is tolerant toabiotic stresses has the ability to endure abiotic stress withoutserious consequences for growth, appearance and yield.

Uniformity. Uniformity, as used herein, describes the similarity betweenplants or plant characteristics which can be a described by qualitativeor quantitative measurements.

Variety. A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties ofplants).

Yield (Tomato yield Tons/Acre). The yield in tons/acre is the actualyield of the tomato fruit at harvest

Tomato Plants

Practically speaking, all cultivated and commercial forms of tomatobelong to a species most frequently referred to as Lycopersiconesculentum Miller. Lycopersicon is a relatively small genus within theextremely large and diverse family Solanaceae which is considered toconsist of around 90 genera, including pepper, tobacco and eggplant. Thegenus Lycopersicon has been divide into two subgenera, the esculentumcomplex which contains those species that can easily be crossed with thecommercial tomato and the peruvianum complex which contains thosespecies which are crossed with considerable difficulty (Stevens, M., andRick, C. M. 1986. Genetics and Breeding. In: The Tomato Crop. Ascientific basis for improvement, pp. 35-109. Atherton, J., Rudich, G.(eds.). Chapman and Hall, New York). Due to its value as a crop, L.esculentum Miller has become widely disseminated all over the world.Even if the precise origin of the cultivated tomato is still somewhatunclear, it seems to come from the Americas, being native to Ecuador,Peru and the Galapagos Island and initially cultivated by Aztecs andIncas as early as 700 AD. Mexico appears to have been the site ofdomestication and the source of the earliest introduction.

It is supposed that the cherry tomato, L. esculentum var. cerasiforme,is the direct ancestor of modern cultivated forms.

Tomato is grown for its fruit, widely used as a fresh market orprocessed product. As a crop, tomato is grown commercially whereverenvironmental conditions permit the production of an economically viableyield. In California, the first largest processing tomato market andsecond largest fresh market in the United States, processing tomato areharvested by machine. The majority of fresh market tomatoes areharvested by hand at vine ripe and mature green stage of ripeness. Freshmarket tomatoes are available in the United States year round.Processing tomato season in California is from late June to October.Processing tomato are used in many forms, as canned tomatoes, tomatojuice, tomato sauce, puree, paste or even catsup. Over the 500,000 acresof tomatoes that are grown annually in the US, approximately 40% aregrown for fresh market consumption, the balance are grown forprocessing.

Tomato is a normally simple diploid species with twelve pairs ofdifferentiated chromosomes. However, polyploidy tomato is also part ofthe present invention. The cultivated tomato is self-fertile and almostexclusively self-pollinating. The tomato flowers are hermaphrodites.Commercial cultivars were initially open pollinated. Most have now beenreplaced by better yielding hybrids. Due to its wide dissemination andhigh value, tomato has been intensively bred. This explains why such awide array of tomato is now available. The shape may range from small tolarge, and there are cherry, plum, pear, blocky, round, and beefsteaktypes. Tomatoes may be grouped by the amount of time it takes for theplants to mature fruit for harvest and, in general, the cultivars areconsidered to be early, midseason or late-maturing. Tomatoes can also begrouped by the plant's growth habit; determinate or indeterminate.Determinate plants tend to grow their foliage first, then set flowersthat mature into fruit if pollination is successful. All of the fruitstend to ripen on a plant at about the same time. Indeterminate tomatoesstart out by growing some foliage, then continue to produce foliage andflowers throughout the growing season. These plants will tend to havetomato fruit in different stages of maturity at any given time. Morerecent developments in tomato breeding have led to a wider array offruit color. In addition to the standard red ripe color, tomatoes can becreamy white, lime green, pink, yellow, golden, orange or purple.

Hybrid vigor has been documented in tomatoes and hybrids are gainingmore and more popularity amongst farmers with uniformity of plantcharacteristics.

Hybrid commercial tomato seed can be produced by hand pollination.Pollen of the male parent is harvested and manually applied to thestigmatic surface of the female inbred. Prior to and after handpollination, flowers are covered so that insects do not bring foreignpollen and create a mix or impurity. Flowers are tagged to identifypollinated fruit from which seed will be harvested.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm.

In tomato, these important traits may include increased fruit number,fruit size and fruit weight, higher seed yield, improved color,resistance to diseases and insects, tolerance to drought and heat,better uniformity, higher nutritional value and better agronomicquality, growth rate, high seed germination, seedling vigor, early fruitmaturity, ease of fruit setting, adaptability for soil and climateconditions, firmness, content in soluble solids, acidity and viscosity.With mechanical harvesting of processing tomato, fruit settingconcentration, harvestability and field holding are also very important.

In some embodiments, particularly desirable traits that may beincorporated by this invention are improved resistance to differentviral, fungal, and bacterial pathogens. Important diseases include butare not limited to Tomato yellow leaf curl virus, Tomato spot wiltvirus, etc. Improved resistance to insect pests is another desirabletrait that may be incorporated into new tomato plants developed by thisinvention. Insect pests affecting the various species of tomato include,but not limited to arthropod pests such as tuta absoluta, franklienellaoccidentalis, bemisia tabaci, etc.

Other desirable traits include traits related to improved tomato fruits.A non-limiting list of fruit phenotypes used during breeding selectioninclude:

-   -   Average of juice bostwick. The juice Bostwick a measurement of        the viscosity. The viscosity or consistency of tomato products        is affected by the degree of concentration of the tomato, the        amount of and extent of degradation of pectin, the size, shape        and quality of the pulp, and probably to a lesser extent, by the        proteins, sugars and other soluble constituents. The viscosity        is measured in Bostwick centimeters by using instruments such as        a Bostwick Consistometer.    -   pH. The pH is a measure of acidity of the fruit puree. A pH        under 4.5 is desirable to prevent bacterial spoilage of finished        products. pH rises as fruit matures.    -   Fruit color. Fruit color is measured as Hunters a/b ratio, where        a represents red/green, positive values are red, negative values        are green and 0 is neutral; b represents yellow/blue, where        positive values are yellow, negative values are blue and 0 is        neutral; a/b represents the intense of redness: large value        represents deep red color, small value represents light or        yellowish red color.    -   Fruit Weight. The weight of a single fruit or the average of        many fruit measured at harvest maturity and recorded in a        convenient unit of measure.    -   Ostwald. The Ostwald is a measurement of serum viscosity whereas        the measurement are taken using an Ostwald viscometer. The serum        is the non-solid portion of a tomato extract after        centrifugation of the tomato puree. The serum viscosity is        affected by the quantity and quality of soluble pectin. Higher        number reflect higher viscosity of the tomato serum.    -   Fruit firmness. The fruit firmness is the resistance to        penetration and is measured using a Digital Durometer Model        DD-4-00 (Rex Gauge Company, Buffalo Grove, Ill., USA). Durometer        readings are taken at 4 locations (each about 90 degrees apart)        on the approximate mid-point of a tomato, with the tomato laying        on its side. From a fruit sample collected at a given location,        the resistance to penetration is measured with the durometer        from 9 individual fruit at 4 locations per fruit (a total of 36        independent measurements). The P5 value is calculated from the        following equation: D-39/10, where D is the value from the        Durometer.        Tomato Breeding

The goal of tomato breeding is to develop new, unique and superiortomato inbred lines and hybrids. The breeder initially selects andcrosses two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. Another method usedto develop new, unique and superior tomato inbred lines and hybridsoccurs when the breeder selects and crosses two or more parental linesfollowed by haploid induction and chromosome doubling that result in thedevelopment of dihaploid inbred lines. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations and the same is true for the utilization of thedihaploid breeding method.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred linesdeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting lines he develops, except possibly ina very gross and general fashion. This unpredictability results in theexpenditure of large research monies to develop superior new tomatoinbred lines and hybrids.

The development of commercial tomato hybrids requires the development ofhomozygous inbred lines, the crossing of these lines, and the evaluationof the hybrid crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop inbred lines from breeding populations. Breeding programscombine desirable traits from two or more inbred lines or variousbroad-based sources into breeding pools from which inbred lines aredeveloped by selfing and selection of desired phenotypes or through thedihaploid breeding method followed by the selection of desiredphenotypes. The new inbreds are crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which havecommercial potential.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

i Pedigree Selection

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents possessing favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). The dihaploid breedingmethod could also be used. Selection of the best individuals is usuallybegun in the F₂ population; then, beginning in the F₃, the bestindividuals in the best families are selected. Replicated testing offamilies, or hybrid combinations involving individuals of thesefamilies, often follows in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potential use asparents of new hybrid cultivars. Similarly, the development of newinbred lines through the dihaploid system requires the selection of thebest inbreds followed by two to five years of testing in hybridcombinations in replicated plots.

ii Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F2 to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F2 individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F2 plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or morefruit containing seed from each plant in a population and blend themtogether to form a bulk seed lot. Part of the bulked seed is used toplant the next generation and part is put in reserve. The procedure hasbeen referred to as modified single-seed descent or the bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster than removing one seed from each fruit by handfor the single seed procedure. The multiple-seed procedure also makes itpossible to plant the same number of seeds of a population eachgeneration of inbreeding. Enough seeds are harvested to make up forthose plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son, pp. 115-161; N. W. Simmonds, 1979, Principles of CropImprovement, Longman Group Limited; W. R. Fehr, 1987, Principles of CropDevelopment, Macmillan Publishing Co.; N. F. Jensen, 1988, PlantBreeding Methodology, John Wiley & Sons).

When the term hybrid tomato plant is used in the context of the presentinvention, this also includes any hybrid tomato plant where one or moredesired trait has been introduced through backcrossing methods, whethersuch trait is a naturally occurring one, a mutant or a transgenic one.Backcrossing methods can be used with the present invention to improveor introduce one or more characteristic into the inbred parental line ofthe hybrid tomato plant of the present invention. The term“backcrossing” as used herein refers to the repeated crossing of ahybrid progeny back to the recurrent parent, i.e., backcrossing one,two, three, four, five, six, seven, eight, nine, or more times to therecurrent parent. The parental tomato plant which contributes the geneor the genes for the desired characteristic is termed the nonrecurrentor donor parent. This terminology refers to the fact that thenonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental tomato plant to which the gene orgenes from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol.

In a typical backcross protocol, the original inbred of interest(recurrent parent) is crossed to a second inbred (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a tomato plant isobtained wherein all the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, generally determined at a 5% significance level when grown in thesame environmental conditions, in addition to the gene or genestransferred from the nonrecurrent parent. It has to be noted that some,one, two, three or more, self-pollination and growing of populationmight be included between two successive backcrosses. Indeed, anappropriate selection in the population produced by theself-pollination, i.e. selection for the desired trait and physiologicaland morphological characteristics of the recurrent parent might beequivalent to one, two or even three additional backcrosses in acontinuous series without rigorous selection, saving then time, moneyand effort to the breeder. A non-limiting example of such a protocolwould be the following: a) the first generation F1 produced by the crossof the recurrent parent A by the donor parent B is backcrossed to parentA, b) selection is practiced for the plants having the desired trait ofparent B, c) selected plant are self-pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,five, six, seven, eight, nine, or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and the physiological and morphological characteristics of parent A.Step (c) may or may not be repeated and included between the backcrossesof step (d).

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred parental line in order to find it then in the hybridmade thereof. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass visual inspection, simplecrossing, but also follow up of the characteristic(s) throughgenetically associated markers and molecular assisted breeding tools.For example, selection of progeny containing the transferred trait isdone by direct selection, visual inspection for a trait associated witha dominant allele, while the selection of progeny for a trait that istransferred via a recessive allele, such as the orange fruit colorcharacteristic in tomato, requires selfing the progeny to determinewhich plant carries the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new parental inbred of a hybridtomato plant according to the invention but that can be improved bybackcrossing techniques. Single gene traits may or may not betransgenic. Examples of these traits include but are not limited to,male sterility (such as the ms1, ms2, ms3, ms4 or ms5 genes), herbicideresistance (such as bar or PAT genes), resistance for bacterial, fungal(genes Cf for resistance to Cladosporium fulvum), or viral disease (geneTy for resistance to Tomato Yellow Leaf Curl Virus (TYLCV), genes Tm-1,Tm-2 and Tm2² for the resistance to the tomato mosaic tobamovirus(ToMV)), insect resistance (gene Mi for resistance to nematodes),increased brix by introduction of specific alleles such as the hir4allele from lycopersicon hirsutum, high lycopene by using the dg mutantas described in U.S. Ser. No. 10/587,789, improved shelf life by usingmutants such as the rin (ripening inhibitor), nor (non-ripening) or cnr(colorless non ripening) alleles, increased firmness or slower softeningof the fruits due, for example in a mutation in an expansin gene,absence of gel (i.e. fruits having a cavity area which is solid andlacks a gel or liquid content male) by the use of the PSAF allele,fertility, enhanced nutritional quality, enhanced sugar content, yieldstability and yield enhancement. These genes are generally inheritedthrough the nucleus. Some known exceptions to this are the genes formale sterility, some of which are inherited cytoplasmically, but stillact as single gene traits. Several of these single gene traits aredescribed in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.

In 1981, the backcross method of breeding counted for 17% of the totalbreeding effort for inbred line development in the United States,accordingly to, Hallauer, A. R. et al. (1988) “Corn Breeding” Corn andCorn Improvement, No. 18, pp. 463-481.

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R. W. Allard's 1960 book,published by John Wiley & Sons, Inc., Principles of Plant Breeding). Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a parental line of a hybrid variety with exactly theadaptation, yielding ability and quality characteristics of therecurrent parent but superior to that parent in the particularcharacteristic(s) for which the improvement program was undertaken.Therefore, this method provides the plant breeder with a high degree ofgenetic control of his work.

The method is scientifically exact because the morphological andagricultural features of the improved variety could be described inadvance and because the same variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method”, 1930 Jour. Amer. Soc.Agron., 22: 289-244).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will bemodified only with regards to genes being transferred, which aremaintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from ‘Hope’ wheat to ‘Bart wheat’ and even pursuing thebackcrosses with the transfer of bunt resistance to create ‘Bart 38’,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in California Commonalfalfa to create ‘Caliverde’. This new ‘Caliverde’ variety producedthrough the backcross process is indistinguishable from CaliforniaCommon except for its resistance to the three named diseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,color characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, ‘Calady’, has been produced by Jones andDavis. As dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e. grain size. ‘Lady Wright’, along grain variety was used as the donor parent and ‘Coloro’, a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety ‘Calady’ was produced.

iii Open-Pollinated Populations

The improvement of open-pollinated populations of such crops as rye,many maizes and sugar beets, herbage grasses, legumes such as alfalfaand clover, and tropical tree crops such as cacao, coconuts, oil palmand some rubber, depends essentially upon changing gene-frequenciestowards fixation of favorable alleles while maintaining a high (but farfrom maximal) degree of heterozygosity.

Uniformity in such populations is impossible and trueness-to-type in anopen-pollinated variety is a statistical feature of the population as awhole, not a characteristic of individual plants. Thus, theheterogeneity of open-pollinated populations contrasts with thehomogeneity (or virtually so) of inbred lines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes to flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement.

First, there is the situation in which a population is changed en masseby a chosen selection procedure. The outcome is an improved populationthat is indefinitely propagable by random-mating within itself inisolation.

Second, the synthetic variety attains the same end result as populationimprovement, but is not itself propagable as such; it has to bereconstructed from parental lines or clones. These plant breedingprocedures for improving open-pollinated populations are well known tothose skilled in the art and comprehensive reviews of breedingprocedures routinely used for improving cross-pollinated plants areprovided in numerous texts and articles, including: Allard, Principlesof Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds, Principlesof Crop Improvement, Longman Group Limited (1979); Hallauer and Miranda,Quantitative Genetics in Maize Breeding, Iowa State University Press(1981); and, Jensen, Plant Breeding Methodology, John Wiley & Sons, Inc.(1988).

A) Mass Selection

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued. In massselection, desirable individual plants are chosen, harvested, and theseed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

B) Synthetics

A synthetic variety is produced by crossing inter se a number ofgenotypes selected for good combining ability in all possible hybridcombinations, with subsequent maintenance of the variety by openpollination. Whether parents are (more or less inbred) seed-propagatedlines, as in some sugar beet and beans (Vicia) or clones, as in herbagegrasses, clovers and alfalfa, makes no difference in principle. Parentsare selected on general combining ability, sometimes by test crosses ortoperosses, more generally by polycrosses. Parental seed lines may bedeliberately inbred (e.g. by selfing or sib crossing). However, even ifthe parents are not deliberately inbred, selection within lines duringline maintenance will ensure that some inbreeding occurs. Clonal parentswill, of course, remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or more cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enters a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

iv. Hybrids

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugarbeet, sunflower andbroccoli. Hybrids can be formed in a number of different ways, includingby crossing two parents directly (single cross hybrids), by crossing asingle cross hybrid with another parent (three-way or triple crosshybrids), or by crossing two different hybrids (four-way or double crosshybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

Hybrid commercial tomato seed is produced by hand pollination. Pollen ofthe male parent is harvested and manually applied to the stigmaticsurface of the female inbred. Prior to, and after hand pollination,flowers are covered so that insects do not bring foreign pollen andcreate a mix or impurity. Flowers are tagged to identify pollinatedfruit from which seed will be harvested.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F1progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F1 hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor and uniformity exhibited byF1 hybrids is lost in the next generation (F2). Consequently, seed fromF2 hybrid varieties is not used for planting stock.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

v. Bulk Segregation Analysis (BSA)

BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, isa method described by Michelmore et al. (Michelmore et al., 1991,Identification of markers linked to disease-resistance genes by bulkedsegregant analysis: a rapid method to detect markers in specific genomicregions by using segregating populations. Proceedings of the NationalAcademy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie etal., 1999, Journal of Experimental Botany, 50(337): 1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F2, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to virus), and theother from the individuals having reversed phenotype (e.g., susceptibleto virus), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs). Markers linked to thephenotype can be identified and used for breeding or QTL mapping.

vi. Hand-Pollination Method

Hand pollination describes the crossing of plants via the deliberatefertilization of female ovules with pollen from a desired male parentplant. In some embodiments the donor or recipient female parent and thedonor or recipient male parent line are planted in the same field. Theinbred male parent can be planted earlier than the female parent toensure adequate pollen supply at the pollination time. In someembodiments, the male parent and female parent can be planted at a ratioof 1 male parent to 4-10 female parents. The male parent may be plantedat the top of the field for efficient male flower collection duringpollination. Pollination is started when the female parent flower isready to be fertilized. Female flower buds that are ready to open in thefollowing days are identified, covered with paper cups or small paperbags that prevent bee or any other insect from visiting the femaleflowers, and marked with any kind of material that can be easily seenthe next morning. In some embodiments, this process is best done in theafternoon. The male flowers of the male parent are collected in theearly morning before they are open and visited by pollinating insects.The covered female flowers of the female parent, which have opened, areun-covered and pollinated with the collected fresh male flowers of themale parent, starting as soon as the male flower sheds pollen. Thepollinated female flowers are again covered after pollination to preventbees and any other insects visit. The pollinated female flowers are alsomarked. The marked fruits are harvested. In some embodiments, the malepollen used for fertilization has been previously collected and stored.

vii. Bee-Pollination Method

Using the bee-pollination method, the parent plants are usually plantedwithin close proximity. In some embodiments more female plants areplanted to allow for a greater production of seed. Breeding of dioeciousspecies can also be done by growing equal amount of each parent plant.Beehives are placed in the field for transfer of pollen by bees from themale parent to the female flowers of the female parent. In someembodiments, fruits set after the introduction of the beehives can bemarked for later collection.

viii. Targeting Induced Local Lesions in Genomes (TILLING)

Breeding schemes of the present application can include crosses withTILLING® plant lines. TILLING® is a method in molecular biology thatallows directed identification of mutations in a specific gene. TILLING®was introduced in 2000, using the model plant Arabidopsis thaliana.TILLING® has since been used as a reverse genetics method in otherorganisms such as zebrafish, corn, wheat, rice, soybean, tomato andlettuce.

The method combines a standard and efficient technique of mutagenesiswith a chemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with asensitive DNA screening-technique that identifies single base mutations(also called point mutations) in a target gene. EcoTILLING is a methodthat uses TILLING® techniques to look for natural mutations inindividuals, usually for population genetics analysis (see Comai, etal., 2003 The Plant Journal 37, 778-786; Gilchrist et al. 2006 Mol.Ecol. 15, 1367-1378; Mejlhede et al. 2006 Plant Breeding 125, 461-467;Nieto et al. 2007 BMC Plant Biology 7, 34-42, each of which isincorporated by reference hereby for all purposes). DEco-TILLING is amodification of TILLING® and EcoTILLING which uses an inexpensive methodto identify fragments (Garvin et al., 2007, DEco-TILLING: An inexpensivemethod for SNP discovery that reduces ascertainment bias. MolecularEcology Notes 7, 735-746).

The TILLING® method relies on the formation of heteroduplexes that areformed when multiple alleles (which could be from a heterozygote or apool of multiple homozygotes and heterozygotes) are amplified in a PCR,heated, and then slowly cooled. A “bubble” forms at the mismatch of thetwo DNA strands (the induced mutation in TILLING® or the naturalmutation or SNP in EcoTILLING), which is then cleaved by single strandednucleases. The products are then separated by size on several differentplatforms.

Several TILLING® centers exists over the world that focus onagriculturally important species: UC Davis (USA), focusing on Rice;Purdue University (USA), focusing on Maize; University of BritishColumbia (CA), focusing on Brassica napus; John Innes Centre (UK),focusing on Brassica rapa; Fred Hutchinson Cancer Research, focusing onArabidopsis; Southern Illinois University (USA), focusing on Soybean;John Innes Centre (UK), focusing on Lotus and Medicago; and INRA(France), focusing on Pea and Tomato.

More detailed description on methods and compositions on TILLING® can befound in U.S. Pat. No. 5,994,075, US 2004/0053236 A1, WO 2005/055704,and WO 2005/048692, each of which is hereby incorporated by referencefor all purposes.

Thus in some embodiments, the breeding methods of the present disclosureinclude breeding with one or more TILLING plant lines with one or moreidentified mutations.

viii Mutation Breeding

Mutation breeding is another method of introducing new traits intotomato plants. Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means or mutating agents including temperature, long-term seedstorage, tissue culture conditions, radiation (such as X-rays, Gammarays, neutrons, Beta radiation, or ultraviolet radiation), chemicalmutagens (such as base analogs like 5-bromo-uracil), antibiotics,alkylating agents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in W. R. Fehr, 1993, Principles ofCultivar Development, Macmillan Publishing Co.

New breeding techniques such as the ones involving the uses of ZincFinger Nucleases or oligonucleotide directed mutagenesis shall also beused to generate genetic variability and introduce new traits intotomato varieties.

ix. Double Haploids and Chromosome Doubling

One way to obtain homozygous plants without the need to cross twoparental lines followed by a long selection of the segregating progeny,and/or multiple back-crossings is to produce haploids and then doublethe chromosomes to form doubled haploids. Haploid plants can occurspontaneously, or may be artificially induced via chemical treatments orby crossing plants with inducer lines (Seymour et al. 2012, PNAS vol.109, pg. 4227-4232; Zhang et al., 2008 Plant Cell Rep. December 27(12)1851-60). The production of haploid progeny can occur via a variety ofmechanisms which can affect the distribution of chromosomes duringgamete formation. The chromosome complements of haploids sometimesdouble spontaneously to produce homozygous doubled haploids (DHs).Mixoploids, which are plants which contain cells having differentploidies, can sometimes arise and may represent plants that areundergoing chromosome doubling so as to spontaneously produce doubledhaploid tissues, organs, shoots, floral parts or plants. Another commontechnique is to induce the formation of double haploid plants with achromosome doubling treatment such as colchicine (El-Hennawy et al.,2011 Vol 56, issue 2 pg. 63-72; Doubled Haploid Production in CropPlants 2003 edited by Maluszynski ISBN 1-4020-1544-5). The production ofdoubled haploid plants yields highly uniform inbred lines and isespecially desirable as an alternative to sexual inbreeding oflonger-generation crops. By producing doubled haploid progeny, thenumber of possible gene combinations for inherited traits is moremanageable. Thus, an efficient doubled haploid technology cansignificantly reduce the time and the cost of inbred and cultivardevelopment.

x. Protoplast Fusion

In another method for breeding plants, protoplast fusion can also beused for the transfer of trait-conferring genomic material from a donorplant to a recipient plant. Protoplast fusion is an induced orspontaneous union, such as a somatic hybridization, between two or moreprotoplasts (cells of which the cell walls are removed by enzymatictreatment) to produce a single bi- or multi-nucleate cell. The fusedcell that may even be obtained with plant species that cannot beinterbred in nature is tissue cultured into a hybrid plant exhibitingthe desirable combination of traits.

xi. Embryo Rescue

Alternatively, embryo rescue may be employed in the transfer ofresistance-conferring genomic material from a donor plant to a recipientplant. Embryo rescue can be used as a procedure to isolate embryo's fromcrosses wherein plants fail to produce viable seed. In this process, thefertilized ovary or immature seed of a plant is tissue cultured tocreate new plants (see Pierik, 1999, In vitro culture of higher plants,Springer, ISBN 079235267x, 9780792352679, which is incorporated hereinby reference in its entirety).

Breeding Evaluation

Each breeding program can include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for use as parents in new commercial cultivars; those stilldeficient in a few traits may be used as parents to produce newpopulations for further selection.

In one embodiment, the plants are selected on the basis of one or morephenotypic traits. Skilled persons will readily appreciate that suchtraits include any observable characteristic of the plant, including forexample growth rate, height, weight, color, taste, smell, changes in theproduction of one or more compounds by the plant (including for example,metabolites, proteins, drugs, carbohydrates, oils, and any othercompounds).

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

It should be appreciated that in certain embodiments, plants may beselected based on the absence, suppression or inhibition of a certainfeature or trait (such as an undesirable feature or trait) as opposed tothe presence of a certain feature or trait (such as a desirable featureor trait).

Selecting plants based on genotypic information is also envisaged (forexample, including the pattern of plant gene expression, genotype, orpresence of genetic markers). Where the presence of one or more geneticmarker is assessed, the one or more marker may already be known and/orassociated with a particular characteristic of a plant; for example, amarker or markers may be associated with an increased growth rate ormetabolite profile. This information could be used in combination withassessment based on other characteristics in a method of the disclosureto select for a combination of different plant characteristics that maybe desirable. Such techniques may be used to identify novel quantitativetrait loci (QTLs). By way of example, plants may be selected based ongrowth rate, size (including but not limited to weight, height, leafsize, stem size, branching pattern, or the size of any part of theplant), general health, survival, tolerance to adverse physicalenvironments and/or any other characteristic, as described hereinbefore.

Further non-limiting examples include selecting plants based on: speedof seed germination; quantity of biomass produced; increased root,and/or leaf/shoot growth that leads to an increased yield (herbage orgrain or fiber or oil) or biomass production; effects on plant growththat results in an increased seed yield for a crop; effects on plantgrowth which result in an increased fruit yield; effects on plant growththat lead to an increased resistance or tolerance disease includingfungal, viral or bacterial diseases or to pests such as insects, mitesor nematodes in which damage is measured by decreased foliar symptomssuch as the incidence of bacterial or fungal lesions, or area of damagedfoliage or reduction in the numbers of nematode cysts or galls on plantroots, or improvements in plant yield in the presence of such plantpests and diseases; effects on plant growth that lead to increasedmetabolite yields; effects on plant growth that lead to improvedaesthetic appeal which may be particularly important in plants grown fortheir form, color or taste, for example the color intensity of tomatoflesh, or the taste of said fruit.

Molecular Breeding Evaluation Techniques

Selection of plants based on phenotypic or genotypic information may beperformed using techniques such as, but not limited to: high through-putscreening of chemical components of plant origin, sequencing techniquesincluding high through-put sequencing of genetic material, differentialdisplay techniques (including DDRT-PCR, and DD-PCR), nucleic acidmicroarray techniques, RNA-seq (Whole Transcriptome Shotgun Sequencing),qRTPCR (quantitative real time PCR).

In one embodiment, the evaluating step of a plant breeding programinvolves the identification of desirable traits in progeny plants.Progeny plants can be grown in, or exposed to conditions designed toemphasize a particular trait (e.g. drought conditions for droughttolerance, lower temperatures for freezing tolerant traits). Progenyplants with the highest scores for a particular trait may be used forsubsequent breeding steps.

In some embodiments, plants selected from the evaluation step canexhibit a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120% or more improvement in aparticular plant trait compared to a control plant.

In other embodiments, the evaluating step of plant breeding comprisesone or more molecular biological tests for genes or other markers. Forexample, the molecular biological test can involve probe hybridizationand/or amplification of nucleic acid (e.g., measuring nucleic aciddensity by Northern or Southern hybridization, PCR) and/or immunologicaldetection (e.g., measuring protein density, such as precipitation andagglutination tests, ELISA (e.g., Lateral Flow test or DAS-ELISA),Western blot, RIA, immune labeling, immunosorbent electron microscopy(ISEM), and/or dot blot).

The procedure to perform a nucleic acid hybridization, an amplificationof nucleic acid (e.g., RT-PCR) or an immunological detection (e.g.,precipitation and agglutination tests, ELISA (e.g., Lateral Flow test orDAS-ELISA), Western blot, RIA, immunogold or immunofluorescent labeling,immunosorbent electron microscopy (ISEM), and/or dot blot tests) areperformed as described elsewhere herein and well-known by one skilled inthe art.

In one embodiment, the evaluating step comprises PCR (semi-quantitativeor quantitative), wherein primers are used to amplify one or morenucleic acid sequences of a desirable gene, or a nucleic acid associatedwith said gene or a desirable trait (e.g., a co-segregating nucleicacid, or other marker).

In another embodiment, the evaluating step comprises immunologicaldetection (e.g., precipitation and agglutination tests, ELISA (e.g.,Lateral Flow test or DAS-ELISA), Western blot, RIA, immuno labeling(gold, fluorescent, or other detectable marker), immunosorbent electronmicroscopy (ISEM), and/or dot blot), wherein one or more gene ormarker-specific antibodies are used to detect one or more desirableproteins. In one embodiment, said specific antibody is selected from thegroup consisting of polyclonal antibodies, monoclonal antibodies,antibody fragments, and combination thereof.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be utilizedin the present disclosure to determine expression of a gene to assistduring the selection step of a breeding scheme. It is a variant ofpolymerase chain reaction (PCR), a laboratory technique commonly used inmolecular biology to generate many copies of a DNA sequence, a processtermed “amplification”. In RT-PCR, however, RNA strand is first reversetranscribed into its DNA complement (complementary DNA, or cDNA) usingthe enzyme reverse transcriptase, and the resulting cDNA is amplifiedusing traditional or real-time PCR.

RT-PCR utilizes a pair of primers, which are complementary to a definedsequence on each of the two strands of the cDNA. These primers are thenextended by a DNA polymerase and a copy of the strand is made after eachcycle, leading to logarithmic amplification.

RT-PCR includes three major steps. The first step is the reversetranscription (RT) where RNA is reverse transcribed to cDNA using areverse transcriptase and primers. This step is very important in orderto allow the performance of PCR since DNA polymerase can act only on DNAtemplates. The RT step can be performed either in the same tube with PCR(one-step PCR) or in a separate one (two-step PCR) using a temperaturebetween 40° C. and 50° C., depending on the properties of the reversetranscriptase used.

The next step involves the denaturation of the dsDNA at 95° C., so thatthe two strands separate and the primers can bind again at lowertemperatures and begin a new chain reaction. Then, the temperature isdecreased until it reaches the annealing temperature which can varydepending on the set of primers used, their concentration, the probe andits concentration (if used), and the cations concentration. The mainconsideration, of course, when choosing the optimal annealingtemperature is the melting temperature (Tm) of the primers and probes(if used). The annealing temperature chosen for a PCR depends directlyon length and composition of the primers. This is the result of thedifference of hydrogen bonds between A-T (2 bonds) and G-C (3 bonds). Anannealing temperature about 5 degrees below the lowest Tm of the pair ofprimers is usually used.

The final step of PCR amplification is the DNA extension from theprimers which is done by the thermostable Taq DNA polymerase usually at72° C., which is the optimal temperature for the polymerase to work. Thelength of the incubation at each temperature, the temperaturealterations and the number of cycles are controlled by a programmablethermal cycler. The analysis of the PCR products depends on the type ofPCR applied. If a conventional PCR is used, the PCR product is detectedusing agarose gel electrophoresis and ethidium bromide (or other nucleicacid staining).

Conventional RT-PCR is a time-consuming technique with importantlimitations when compared to real time PCR techniques. This, combinedwith the fact that ethidium bromide has low sensitivity, yields resultsthat are not always reliable. Moreover, there is an increasedcross-contamination risk of the samples since detection of the PCRproduct requires the post-amplification processing of the samples.Furthermore, the specificity of the assay is mainly determined by theprimers, which can give false-positive results. However, the mostimportant issue concerning conventional RT-PCR is the fact that it is asemi or even a low quantitative technique, where the amplicon can bevisualized only after the amplification ends.

Real time RT-PCR provides a method where the amplicons can be visualizedas the amplification progresses using a fluorescent reporter molecule.There are three major kinds of fluorescent reporters used in real timeRT-PCR, general non-specific DNA Binding Dyes such as SYBR Green I,TaqMan Probes and Molecular Beacons (including Scorpions).

The real time PCR thermal cycler has a fluorescence detection threshold,below which it cannot discriminate the difference between amplificationgenerated signal and background noise. On the other hand, thefluorescence increases as the amplification progresses and theinstrument performs data acquisition during the annealing step of eachcycle. The number of amplicons will reach the detection baseline after aspecific cycle, which depends on the initial concentration of the targetDNA sequence. The cycle at which the instrument can discriminate theamplification generated fluorescence from the background noise is calledthe threshold cycle (Ct). The higher is the initial DNA concentration,the lower its Ct will be.

Other forms of nucleic acid detection can include next generationsequencing methods such as DNA SEQ or RNA SEQ using any known sequencingplatform including, but not limited to: Roche 454, Solexa GenomeAnalyzer, AB SOLiD, Illumina GA/HiSeq, Ion PGM, Mi Seq, among others(Liu et al., 2012 Journal of Biomedicine and Biotechnology Volume 2012ID 251364; Franca et al., 2002 Quarterly Reviews of Biophysics 35 pg.169-200; Mardis 2008 Genomics and Human Genetics vol. 9 pg. 387-402).

In other embodiments, nucleic acids may be detected with other highthroughput hybridization technologies including microarrays, gene chips,LNA probes, nanoStrings, and fluorescence polarization detection amongothers.

In some embodiments, detection of markers can be achieved at an earlystage of plant growth by harvesting a small tissue sample (e.g., branch,or leaf disk). This approach is preferable when working with largepopulations as it allows breeders to weed out undesirable progeny at anearly stage and conserve growth space and resources for progeny whichshow more promise. In some embodiments the detection of markers isautomated, such that the detection and storage of marker data is handledby a machine. Recent advances in robotics have also led to full serviceanalysis tools capable of handling nucleic acid/protein markerextractions, detection, storage and analysis.

Quantitative Trait Loci

Breeding schemes of the present application can include crosses betweendonor and recipient plants. In some embodiments said donor plantscontain a gene or genes of interest which may confer the plant with adesirable phenotype. The recipient line can be an elite line havingcertain favorite traits such for commercial production. In oneembodiment, the elite line may contain other genes that also impart saidline with the desired phenotype. When crossed together, the donor andrecipient plant may create a progeny plant with combined desirable lociwhich may provide quantitatively additive effect of a particularcharacteristic. In that case, QTL mapping can be involved to facilitatethe breeding process.

A QTL (quantitative trait locus) mapping can be applied to determine theparts of the donor plant's genome conferring the desirable phenotype,and facilitate the breeding methods. Inheritance of quantitative traitsor polygenic inheritance refers to the inheritance of a phenotypiccharacteristic that varies in degree and can be attributed to theinteractions between two or more genes and their environment. Though notnecessarily genes themselves, quantitative trait loci (QTLs) arestretches of DNA that are closely linked to the genes that underlie thetrait in question. QTLs can be molecularly identified to help mapregions of the genome that contain genes involved in specifying aquantitative trait. This can be an early step in identifying andsequencing these genes.

Typically, QTLs underlie continuous traits (those traits that varycontinuously, e.g. yield, height, level of resistance to virus, etc.) asopposed to discrete traits (traits that have two or several charactervalues, e.g. smooth vs. wrinkled peas used by Mendel in hisexperiments). Moreover, a single phenotypic trait is usually determinedby many genes. Consequently, many QTLs are associated with a singletrait.

A quantitative trait locus (QTL) is a region of DNA that is associatedwith a particular phenotypic trait—these QTLs are often found ondifferent chromosomes. Knowing the number of QTLs that explainsvariation in the phenotypic trait tells about the genetic architectureof a trait. It may tell that a trait is controlled by many genes ofsmall effect, or by a few genes of large effect.

Another use of QTLs is to identify candidate genes underlying a trait.Once a region of DNA is identified as contributing to a phenotype, itcan be sequenced. The DNA sequence of any genes in this region can thenbe compared to a database of DNA for genes whose function is alreadyknown.

In a recent development, classical QTL analyses are combined with geneexpression profiling i.e. by DNA microarrays. Such expression QTLs(e-QTLs) describes cis- and trans-controlling elements for theexpression of often disease-associated genes. Observed epistatic effectshave been found beneficial to identify the gene responsible by across-validation of genes within the interacting loci with metabolicpathway- and scientific literature databases.

QTL mapping is the statistical study of the alleles that occur in alocus and the phenotypes (physical forms or traits) that they produce(see, Meksem and Kahl, The handbook of plant genome mapping: genetic andphysical mapping, 2005, Wiley-VCH, ISBN 3527311165, 9783527311163).Because most traits of interest are governed by more than one gene,defining and studying the entire locus of genes related to a trait giveshope of understanding what effect the genotype of an individual mighthave in the real world.

Statistical analysis is required to demonstrate that different genesinteract with one another and to determine whether they produce asignificant effect on the phenotype. QTLs identify a particular regionof the genome as containing a gene that is associated with the traitbeing assayed or measured. They are shown as intervals across achromosome, where the probability of association is plotted for eachmarker used in the mapping experiment.

To begin, a set of genetic markers must be developed for the species inquestion. A marker is an identifiable region of variable DNA. Biologistsare interested in understanding the genetic basis of phenotypes(physical traits). The aim is to find a marker that is significantlymore likely to co-occur with the trait than expected by chance, that is,a marker that has a statistical association with the trait. Ideally,they would be able to find the specific gene or genes in question, butthis is a long and difficult undertaking. Instead, they can more readilyfind regions of DNA that are very close to the genes in question. When aQTL is found, it is often not the actual gene underlying the phenotypictrait, but rather a region of DNA that is closely linked with the gene.

For organisms whose genomes are known, one might now try to excludegenes in the identified region whose function is known with somecertainty not to be connected with the trait in question. If the genomeis not available, it may be an option to sequence the identified regionand determine the putative functions of genes by their similarity togenes with known function, usually in other genomes. This can be doneusing BLAST, an online tool that allows users to enter a primarysequence and search for similar sequences within the BLAST database ofgenes from various organisms.

Another interest of statistical geneticists using QTL mapping is todetermine the complexity of the genetic architecture underlying aphenotypic trait. For example, they may be interested in knowing whethera phenotype is shaped by many independent loci, or by a few loci, and dothose loci interact. This can provide information on how the phenotypemay be evolving.

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization is possible due to DNA-DNAhybridization techniques (RFLP) and/or due to techniques using thepolymerase chain reaction (e.g. STS, microsatellites, AFLP). Alldifferences between two parental genotypes will segregate in a mappingpopulation based on the cross of these parental genotypes. Thesegregation of the different markers may be compared and recombinationfrequencies can be calculated. The recombination frequencies ofmolecular markers on different chromosomes are generally 50%. Betweenmolecular markers located on the same chromosome the recombinationfrequency depends on the distance between the markers. A lowrecombination frequency corresponds to a low distance between markers ona chromosome. Comparing all recombination frequencies will result in themost logical order of the molecular markers on the chromosomes. Thismost logical order can be depicted in a linkage map (Paterson, 1996,Genome Mapping in Plants. R. G. Landes, Austin.). A group of adjacent orcontiguous markers on the linkage map that is associated to a reduceddisease incidence and/or a reduced lesion growth rate pinpoints theposition of a QTL.

The nucleic acid sequence of a QTL may be determined by methods known tothe skilled person. For instance, a nucleic acid sequence comprisingsaid QTL or a resistance-conferring part thereof may be isolated from adonor plant by fragmenting the genome of said plant and selecting thosefragments harboring one or more markers indicative of said QTL.Subsequently, or alternatively, the marker sequences (or parts thereof)indicative of said QTL may be used as (PCR) amplification primers, inorder to amplify a nucleic acid sequence comprising said QTL from agenomic nucleic acid sample or a genome fragment obtained from saidplant. The amplified sequence may then be purified in order to obtainthe isolated QTL. The nucleotide sequence of the QTL, and/or of anyadditional markers comprised therein, may then be obtained by standardsequencing methods.

One or more such QTLs associated with a desirable trait in a donor plantcan be transferred to a recipient plant to make incorporate thedesirable train into progeny plants by transferring and/or breedingmethods.

In one embodiment, an advanced backcross QTL analysis (AB-QTL) is usedto discover the nucleotide sequence or the QTLs responsible for theresistance of a plant. Such method was proposed by Tanksley and Nelsonin 1996 (Tanksley and Nelson, 1996, Advanced backcross QTL analysis: amethod for simultaneous discovery and transfer of valuable QTL fromun-adapted germplasm into elite breeding lines. Theor Appl Genet92:191-203) as a new breeding method that integrates the process of QTLdiscovery with variety development, by simultaneously identifying andtransferring useful QTL alleles from un-adapted (e.g., land races, wildspecies) to elite germplasm, thus broadening the genetic diversityavailable for breeding. AB-QTL strategy was initially developed andtested in tomato, and has been adapted for use in other crops includingrice, maize, wheat, pepper, barley, and bean. Once favorable QTL allelesare detected, only a few additional marker-assisted generations arerequired to generate near isogenic lines (NILs) or introgression lines(ILs) that can be field tested in order to confirm the QTL effect andsubsequently used for variety development.

Isogenic lines in which favorable QTL alleles have been fixed can begenerated by systematic backcrossing and introgressing of marker-defineddonor segments in the recurrent parent background. These isogenic linesare referred as near isogenic lines (NILs), introgression lines (ILs),backcross inbred lines (BILs), backcross recombinant inbred lines(BCRIL), recombinant chromosome substitution lines (RCSLs), chromosomesegment substitution lines (CSSLs), and stepped aligned inbredrecombinant strains (STAIRSs). An introgression line in plant molecularbiology is a line of a crop species that contains genetic materialderived from a similar species. ILs represent NILs with relatively largeaverage introgression length, while BILs and BCRILs are backcrosspopulations generally containing multiple donor introgressions per line.As used herein, the term “introgression lines or ILs” refers to plantlines containing a single marker defined homozygous donor segment, andthe term “pre-ILs” refers to lines which still contain multiplehomozygous and/or heterozygous donor segments.

To enhance the rate of progress of introgression breeding, a geneticinfrastructure of exotic libraries can be developed. Such an exoticlibrary comprises of a set of introgression lines, each of which has asingle, possibly homozygous, marker-defined chromosomal segment thatoriginates from a donor exotic parent, in an otherwise homogenous elitegenetic background, so that the entire donor genome would be representedin a set of introgression lines. A collection of such introgressionlines is referred as libraries of introgression lines or IL libraries(ILLs). The lines of an ILL cover usually the complete genome of thedonor, or the part of interest. Introgression lines allow the study ofquantitative trait loci, but also the creation of new varieties byintroducing exotic traits. High resolution mapping of QTL using ILLsenable breeders to assess whether the effect on the phenotype is due toa single QTL or to several tightly linked QTL affecting the same trait.In addition, sub-ILs can be developed to discover molecular markerswhich are more tightly linked to the QTL of interest, which can be usedfor marker-assisted breeding (MAB). Multiple introgression lines can bedeveloped when the introgression of a single QTL is not sufficient toresult in a substantial improvement in agriculturally important traits(Gur and Zamir, Unused natural variation can lift yield barriers inplant breeding, 2004, PLoS Biol.; 2(10):e245).

Plant Transformation

In some embodiments, the present invention provides transformed tomatoplants or parts thereof that have been transformed so that its geneticmaterial contains one or more transgenes, preferably operably linked toone or more regulatory elements. Also, the invention provides methodsfor producing a tomato plant containing in its genetic material one ormore transgenes, preferably operably linked to one or more regulatoryelements, by crossing transformed tomato plants with a second plant ofanother tomato, so that the genetic material of the progeny that resultsfrom the cross contains the transgene(s), preferably operably linked toone or more regulatory elements. The invention also provides methods forproducing a tomato plant that contains in its genetic material one ormore transgene(s), wherein the method comprises crossing a tomato with asecond plant of another tomato which contains one or more transgene(s)operably linked to one or more regulatory element(s) so that the geneticmaterial of the progeny that results from the cross contains thetransgene(s) operably linked to one or more regulatory element(s).Transgenic tomato plants, or parts thereof produced by the method are inthe scope of the present invention.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed hybrid tomato plant HMX4909.

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

i Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985), Jefferson et al., Embo J.3901-390764, (1987), Diant, et al., Molecular Breeding, 3:1, 75-86(1997), Valles et al., PI. Cell. Rep. 145-148:13 (1984). A. tumefaciensand A. rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems andmethods for Agrobacterium-mediated gene transfer are provided by Gruberet al., supra, Miki et al., supra, and Moloney et al., Plant CellReports 8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7,1997.

ii. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has been achieved in rice and corn. Hiei et al., ThePlant Journal 6:271-282 (1994) and U.S. Pat. No. 5,591,616 issued Jan.7, 1997. Several methods of plant transformation, collectively referredto as direct gene transfer, have been developed as an alternative toAgrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 micron. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Russell, D. R.,et al., Pl. Cell. Rep., 12, 165-169 (1993); Aragao, F. J. L., et al.,Plant Mol. Biol., 20, 357-359 (1992); Aragao, Theor. Appl. Genet.,93:142-150 (1996); Kim, J.; Minamikawa, T., Plant Science, 117:131-138(1996); Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J. C.,Trends Biotech. 6:299 (1988), Klein et al., BioTechnology6:559-563(1988), Sanford, J. C., Physiol Plant 7:206 (1990), Klein et al.,BioTechnology 10:268 (1992). Gray et al., Plant Cell Tissue and OrganCulture. 1994, 37:2, 179-184.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., BioTechnology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. D'Halluin et al., Plant Cell 4:1495-1505 (1992) andSpencer et al., Plant Mol. Biol. 24:51-61 (1994).

Any DNA sequence(s), whether from a different species or from the samespecies that is inserted into the genome using transformation isreferred to herein collectively as “transgenes.” In some embodiments ofthe invention, a transformed variant of hybrid tomato HMX4909 maycontain at least one transgene but could contain at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 transgenes. In another embodiment of the invention, atransformed variant of the another tomato plant used as the donor linemay contain at least one transgene but could contain at least 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 transgenes.

Following transformation of tomato target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic inbred line or a transgenic hybrid plant. Thetransgenic inbred line could then be crossed, with another(non-transformed or transformed) inbred line, in order to produce a newtransgenic inbred line or plant. Alternatively, a genetic trait whichhas been engineered into a particular tomato plant using the foregoingtransformation techniques could be moved into another tomato plant usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

iii Selection

For efficient plant transformation, a selection method must be employedsuch that whole plants are regenerated from a single transformed celland every cell of the transformed plant carries the DNA of interest.These methods can employ positive selection, whereby a foreign gene issupplied to a plant cell that allows it to utilize a substrate presentin the medium that it otherwise could not use, such as mannose or xylose(for example, refer U.S. Pat. Nos. 5,767,378; 5,994,629). Moretypically, however, negative selection is used because it is moreefficient, utilizing selective agents such as herbicides or antibioticsthat either kill or inhibit the growth of non-transformed plant cellsand reducing the possibility of chimeras. Resistance genes that areeffective against negative selective agents are provided on theintroduced foreign DNA used for the plant transformation. For example,one of the most popular selective agents used is the antibiotickanamycin, together with the resistance gene neomycin phosphotransferase(nptII), which confers resistance to kanamycin and related antibiotics(see, for example, Messing & Vierra, Gene 19: 259-268 (1982); Bevan etal., Nature 304:184-187 (1983)). However, many different antibiotics andantibiotic resistance genes can be used for transformation purposes(refer U.S. Pat. Nos. 5,034,322, 6,174,724 and 6,255,560). In addition,several herbicides and herbicide resistance genes have been used fortransformation purposes, including the bar gene, which confersresistance to the herbicide phosphinothricin (White et al., Nucl AcidsRes 18: 1062 (1990), Spencer et al., Theor Appl Genet 79: 625-631(1990),U.S. Pat. Nos. 4,795,855, 5,378,824 and 6,107,549). In addition, thedhfr gene, which confers resistance to the anticancer agentmethotrexate, has been used for selection (Bourouis et al., EMBO J.2(7): 1099-1104 (1983).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include beta-glucuronidase (GUS,beta-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984), Valles et al, Plant CellReport 3:3-4 145-148 (1994), Shetty et al., FoodBiotechnology 11:2111-128 (1997)

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are also available. However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers. A gene encoding Green Fluorescent Protein (GFP)has been utilized as a marker for gene expression in prokaryotic andeukaryotic cells. Chalfie et al., Science 263:802 (1994). GFP andmutants of GFP may be used as screenable markers.

iv Expression Vectors

Genes can be introduced in a site directed fashion using homologousrecombination. Homologous recombination permits site-specificmodifications in endogenous genes and thus inherited or acquiredmutations may be corrected, and/or novel alterations may be engineeredinto the genome. Homologous recombination and site-directed integrationin plants are discussed in, for example, U.S. Pat. Nos. 5,451,513;5,501,967 and 5,527,695.

The expression control elements used to regulate the expression of agiven protein can either be the expression control element that isnormally found associated with the coding sequence (homologousexpression element) or can be a heterologous expression control element.A variety of homologous and heterologous expression control elements areknown in the art and can readily be used to make expression units foruse in the present invention. Transcription initiation regions, forexample, can include any of the various opine initiation regions, suchas octopine, mannopine, nopaline and the like that are found in the Tiplasmids of Agrobacterium tumefaciens. Alternatively, plant viralpromoters can also be used, such as the cauliflower mosaic virus 19S and35S promoters (CaMV 19S and CaMV 35S promoters, respectively) to controlgene expression in a plant (U.S. Pat. Nos. 5,352,605; 5,530,196 and5,858,742 for example). Enhancer sequences derived from the CaMV canalso be utilized (U.S. Pat. Nos. 5,164,316; 5,196,525; 5,322,938;5,530,196; 5,352,605; 5,359,142; and 5,858,742 for example). Lastly,plant promoters such as prolifera promoter, fruit specific promoters,Ap3 promoter, heat shock promoters, seed specific promoters, etc. canalso be used.

Either a gamete-specific promoter, a constitutive promoter (such as theCaMV or Nos promoter), an organ-specific promoter (such as the E8promoter from tomato), or an inducible promoter is typically ligated tothe protein or antisense encoding region using standard techniques knownin the art. The expression unit may be further optimized by employingsupplemental elements such as transcription terminators and/or enhancerelements.

Thus, for expression in plants, the expression units will typicallycontain, in addition to the protein sequence, a plant promoter region, atranscription initiation site and a transcription termination sequence.Unique restriction enzyme sites at the 5′ and 3′ ends of the expressionunit are typically included to allow for easy insertion into apre-existing vector.

In the construction of heterologous promoter/structural gene orantisense combinations, the promoter is preferably positioned about thesame distance from the heterologous transcription start site as it isfrom the transcription start site in its natural setting. As is known inthe art, however, some variation in this distance can be accommodatedwithout loss of promoter function.

In addition to a promoter sequence, the expression cassette can alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes. If the mRNA encoded by the structural gene is tobe efficiently processed, DNA sequences which direct polyadenylation ofthe RNA are also commonly added to the vector construct. Polyadenylationsequences include, but are not limited to the Agrobacterium octopinesynthase signal (Gielen et al., EMBO J 3:835-846 (1984)) or the nopalinesynthase signal (Depicker et al., Mol. and Appl. Genet. 1:561-573(1982)). The resulting expression unit is ligated into or otherwiseconstructed to be included in a vector that is appropriate for higherplant transformation. One or more expression units may be included inthe same vector. The vector will typically contain a selectable markergene expression unit by which transformed plant cells can be identifiedin culture. Usually, the marker gene will encode resistance to anantibiotic, such as G418, hygromycin, bleomycin, kanamycin, orgentamicin or to an herbicide, such as glyphosate (Round-Up) orglufosinate (BASTA) or atrazine. Replication sequences, of bacterial orviral origin, are generally also included to allow the vector to becloned in a bacterial or phage host; preferably a broad host range forprokaryotic origin of replication is included. A selectable marker forbacteria may also be included to allow selection of bacterial cellsbearing the desired construct. Suitable prokaryotic selectable markersinclude resistance to antibiotics such as ampicillin, kanamycin ortetracycline. Other DNA sequences encoding additional functions may alsobe present in the vector, as is known in the art. For instance, in thecase of Agrobacterium transformations, T-DNA sequences will also beincluded for subsequent transfer to plant chromosomes.

To introduce a desired gene or set of genes by conventional methodsrequires a sexual cross between two lines, and then repeatedback-crossing between hybrid offspring and one of the parents until aplant with the desired characteristics is obtained. This process,however, is restricted to plants that can sexually hybridize, and genesin addition to the desired gene will be transferred.

Recombinant DNA techniques allow plant researchers to circumvent theselimitations by enabling plant geneticists to identify and clone specificgenes for desirable traits, such as improved fatty acid composition, andto introduce these genes into already useful varieties of plants. Oncethe foreign genes have been introduced into a plant, that plant can thenbe used in conventional plant breeding schemes (e.g., pedigree breeding,single-seed-descent breeding schemes, reciprocal recurrent selection) toproduce progeny which also contain the gene of interest.

v Promoters

Genes included in expression vectors must be driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain organs,such as leaves, roots, seeds and tissues such as fibers, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as“tissue-preferred”. Promoters which initiate transcription only incertain tissue are referred to as “tissue-specific”. A “cell type”specific promoter primarily drives expression in certain cell types inone or more organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue-specific, tissue-preferred, cell type specific, andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

A) Inducible Promoters

An inducible promoter is operably linked to a gene for expression intomato. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in tomato. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners (Gatzet al., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10(Gatz et al., Mol. Gen. Genetics 227:229-237 (1991)). A particularlypreferred inducible promoter is a promoter that responds to an inducingagent to which plants do not normally respond. An exemplary induciblepromoter is the inducible promoter from a steroid hormone gene, thetranscriptional activity of which is induced by a glucocorticosteroidhormone (Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991)).

B) Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression intomato or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in tomato.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)). The ALS promoter, XbaI/NcoI fragment 5′ to the Brassicanapus ALS3 structural gene (or a nucleotide sequence similarity to saidXbaI/NcoI fragment), represents a particularly useful constitutivepromoter. See PCT application WO96/30530.C. Tissue-specific orTissue-preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin tomato. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in tomato. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Knox, C., et al.,Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol. 91:124-129(1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell. Biol.108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon, etal., Cell 39:499-509 (1984), Stiefel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a one embodiment, the transgenic plant provided forcommercial production of foreign protein is tomato plant. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology, Glick and Thompson Eds., CRC Press, Boca Raton269:284 (1993). Map information concerning chromosomal location isuseful for proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with one ormore cloned resistance gene to engineer plants that are resistant tospecific pathogen strains. See, for example Jones et al., Science266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomato Ptogene for resistance to Pseudomonas syringae pv. tomato encodes a proteinkinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2 genefor resistance to Pseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btalpha-endotoxin gene. Moreover, DNA molecules encoding alpha-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus alpha-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. Pratt et al., Biochem.Biophys. Res. Comm. 163:1243 (1989) (an allostatin is identified inDiploptera puntata). See also U.S. Pat. No. 5,266,317 to Tomalski etal., who disclose genes encoding insect-specific, paralytic neurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci. 89:43 (1993),of heterologous expression of a cecropin-beta, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-alpha-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-alpha-1, 4-D-galacturonase. See Lamb et al.,BioTechnology 10:1436 (1992). The cloning and characterization of a genewhich encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., BioTechnology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant 5-enolpyruvlshikimate-3phosphate synthase (EPSP) and aroA genes, respectively) and otherphosphono compounds such as glufosinate (phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicus PAT, bar, genes), andpyridinoxy or phenoxy propionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession number 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European patent application No. 0 333 033 to Kumadaet al., and U.S. Pat. No. 4,975,374 to Goodman et al., disclosenucleotide sequences of glutamine synthatase genes which conferresistance to herbicides such as L-phosphinothricin. The nucleotidesequence of a PAT gene is provided in European application No. 0 242 246to Leemans et al. DeGreef et al., BioTechnology 7:61 (1989), describethe production of transgenic plants that express chimeric bar genescoding for PAT activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Accl-S1, Accl-S2 and Accl-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Increased sweetness and flavor of the fruit by introduction of a geneencoding sweet-tasting proteins such as monellin (Penarrubia et al.,Biotechnology. 1992, 10: 5, 561-564) or thaumatin (Bartoszewski et al,Plant Breeding 122, 347-351 (2003)).

B. Reduced ethylene biosynthesis to control ripening by introduction ofan antisense construct of the ACC oxidase into tomato. For example, seeAyub et al, Nature Biotechnology 14: 862 (1996).

C. Delayed senescence and improved ripening control by transferring agene or acting on the transcription of a gene involved in plantsenescence. See Wang et al. in Plant Mol. Bio. 52:1223-1235 (2003) onthe role of the deoxyhypusine synthase in the senescence. See also U.S.Pat. No. 6,538,182 issued Mar. 25, 2003.

D. Improved salt tolerance by transforming tomato plant with HAL 1, ayeast regulatory gene involved in stress tolerance, as shown in Serranoet al., Scientia Horticulturae. 1999, 78: 1/4, 261-269 or in Bordas etal., Transgenic Research. 1997, 6: 1, 41-50.

E. Obtained male sterile plants, especially useful in hybrid tomatoproduction, by introduction of a gene encoding a tobacco PR Glucanase(WO9738116).

F. Increased flooding tolerance, for example by transforming a plantwith a bacterial enzyme ACC deaminase. See Grichko et al., PlantPhysiology and Biochemistry. 2001. 39: 1, 19-25.

G. Improved juice and pulp viscosity, by transforming the plant with anantisense gene of polygalacturonase. For example, see Porretta et al.,Food Chemistry. 1998, 62: 3, 283-290, or Errington et al., Journal ofthe Science of Food and Agriculture, 1998. 76: 4, 515-519.

H. Reduced polyethylene production in order to better control theripening of the fruit, by transforming the plant with aS-adenosylmethionine hydrolase. See Good et al., Plant MolecularBiology. 1994, 26: 3, 781-790.

Tissue Culture

As it is well known in the art, tissue culture of tomato can be used forthe in vitro regeneration of tomato plants. Tissues cultures of varioustissues of tomato and regeneration of plants therefrom are well knownand published. By way of example, a tissue culture comprising organs hasbeen used to produce regenerated plants as described in Girish-Chandelet al., Advances in Plant Sciences. 2000, 13: 1, 11-17, Costa et al.,Plant Cell Report. 2000, 19: 3327-332, Plastira et al., ActaHorticulturae. 1997, 447, 231-234, Zagorska et al., Plant Cell Report.1998, 17: 12 968-973, Asahura et al., Breeding Science. 1995, 45:455-459, Chen et al., Breeding Science. 1994, 44: 3, 257-262, Patil etal., Plant and Tissue and Organ Culture. 1994, 36: 2, 255-258. It isclear from the literature that the state of the art is such that thesemethods of obtaining plants are routinely used and have a very high rateof success. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce tomato plants having thephysiological and morphological characteristics of hybrid tomato plantHMX4909.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, leaves, stems,roots, root tips, anthers, pistils, meristematic cells, axillary buds,ovaries, seed coat, endosperm, hypocotyls, cotyledons and the like.Means for preparing and maintaining plant tissue culture are well knownin the art. By way of example, a tissue culture comprising organs hasbeen used to produce regenerated plants. U.S. Pat. Nos. 5,959,185,5,973,234, and 5,977,445 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

EXAMPLES

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

Example 1 Development of New HMX4909 Tomato Variety

Hybrid tomato plant HMX4909 has superior characteristics. The femaleFTOM4 and male FTOM3 parents were crossed to produce hybrid (F1) seedsof HMX4909. The seeds of HMX4909 can be grown to produce hybrid plantsand parts thereof. The hybrid HMX4909 can be propagated by seeds bycrossing tomato inbred line FTOM4 with tomato inbred line FTOM3 orvegetatively.

The origin and breeding history of hybrid plant HMX4909 can besummarized as follows: the line FTOM4 was used as the female plant andcrossed by pollen from the line FTOM3 (both proprietary lines owned byHM.CLAUSE, Inc.). The first trial planting of this hybrid was done infive different locations (from Fresno County to Yolo County) inCalifornia in the summer of the first year of development. The hybridwas further trialed for two additional years, an example of such trialbeing disclosed in Tables 2 and 3.

The inbred line FTOM4 is a parent with strong dark vine, produces firmdark red fruits, this inbred line was used as female parent in thiscross.

The inbred FTOM3 is a strong plant with dark leaves and strong fieldholding line, it was used as the male parent in this cross.

Hybrid tomato plant HMX4909 is similar to hybrid tomato plant H8504.H8504 is a commercial variety. As shown in tables 2 and 3, while similarto hybrid tomato plant H8504, there are significant differencesincluding the brix raw value of fruits extract which is 5.56 for HMX4909while it is 4.83 for H8504, the average fruit yield for five plantswhich is 93 pounds for HMX4909 while it is 85 for H8504, the averagefruit weight which is 97 grams for HMX4909 while it is 75 grams forH8504, the fruit firmness which is 4.47 for HMX4909 while it is 4.10 forH8504. The fruit bostwick is also different, being 14.52 for HMX4909 and12.77 for H8504. Resistance to viruses are also different, HMX4909 beingresistant to Tomato spotted wilt virus while H8504 is susceptible.Tomato hybrid HMX4909 is resistant to the fungal pathogen which causesfusarium radicii while H8504 is susceptible to fusarium radiciiinfection.

Some of the criteria used to select the hybrid HMX4909 as well as theirinbred parent lines in various generations include: yield, fruit brix,bostwick, Ostwald, fruit weight, fruit firmness, fruit color, diseaseresistance, and earliness.

TABLE 1 Comparison between HMX4885 and H8504 HMX4909 H8504 Observationtrial planted in: Field Field Observation trial planting type:Transplant Transplant Dates of seeding/transplanting: March to May Marchto May Location of trials: Fresno, Kern, Fresno, Kern, King, Yolo, King,Yolo, Stockton, Stockton, Sacramento, Sacramento, California CaliforniaObservation trial planting type: Transplanted and Transplanted unstakedand unstaked Seedling anthocyanin in hypocotyl of 2-15 cm: 1 = absent; 2= present 2 2 habit of 3-4 week old seedling: 1 = normal; 2 = compact 11 Mature plant: height 63.2 cm 58.5 cm growth type: 1 = indeterminate; 2= determinate 2 2 Plant form: 1 = normal; 2 = compact; 3 = dwarf; 4 =brachytic 1 1 size of canopy (compared to others of similar form); 1 =small; 2 = medium; 3 2 3 = large; habit: 1 = sprawling; 2 = semi-erect;3 = erect 2 2 branching: 1 = sparse; 2 = intermediate; 3 = profuse 3 3branching at cotyledon or first leafy node: 1 = present; 2 = absent 2 2number of nodes before first inflorescence 4 to 6 3 to 6 number of nodesbetween early (1st to 2nd, 2nd to 3rd) inflorescence 1 to 2 1 to 2number of nodes between later developing inflorescence 1 to 2 1 to 2pubescence on younger stems: 1 = smooth (no long hairs); 2 = sparselyhairy 2 2 (scattered long hairs); 3 = moderately hairy; 4 = denselyhairy or wooly Leaf type: 1 = tomato; 2 = potato (Trip-L-Crop) 1 1Morphology margins of major leaflets: 1 = absent; 2 = shallowly toothedor scalloped; 2 2 3 = deeply toothed or cut, specially towards basemarginal rolling or wiltiness: 1 = absent; 2 = slight; 3 = moderate; 4 =strong 1 2 onset of leaflet rolling: 1 = early-season; 2 = mid-season; 3= late-season 3 2 surface of major leaflets: 1 = smooth; 2 = rogues(bumpy or veiny) 1 1 pubescence: 1 = smooth (no long hairs); 2 = normal;3 = hirsute; 4 = wooly 2 2 Inflorescence Type: 1 =simple; 2 = forked (2major axes); 3 = compound (much branched) 1 + 2 1 + 2 number of flowersin inflorescence average 6.5 6 leafy or “running” inflorescence: 1 =absent; 2 = occasional; 3 = frequent 1 1 Flower calyx: 1 = normal, lobesawl-shaped; 2 = macrocalyx, lobes large, 3 = fleshy 1 1 calyx-lobes: 1 =shorter than corolla; 2 = approx., equaling corolla; 3 = distinctly 1 1longer than corolla corolla color: 1 = yellow: 2 = old gold; 3 = whiteor tan 1 1 style pubescence: 1 = absent; 2 = sparse; 3 = dense 1 1anthers: 1 = all fused into tube; 2 = separating into 2 or more 1 1Fruit typical shape in longitudinal section date-like date-like shape oftransverse section: 1 = round; 2 = flattened; 3 = angular; 4 = irregular2 2 shape of stem end: 1 = flat; 2 = indented 2 2 shape of blossom end:1 = indented; 2 = flat; 3 = nippled; 4 = tapered 2 3 shape of pistilscar: 1 = dot; 2 = stellate; 3 = linear; 4 = irregular 1 1 abscissionlayer: 1 = present (pedicellate); 2 = absent (jointless) 2 2 point ofdetachment of fruit at harvest: 1 = at pedicel joint; 2 = at calyx 2 2attachment Length of mature fruit (stem axis) 58.3 mm 56.9 mm Diameterof fruit at widest point 52.8 mm 50.1 mm Weight of mature fruit 97.0 g75.0 g Number of locules: 1 = two; 2 = three; 3 = four or five; 4 = morethan 5 1 + 2 1 + 2 Fruit surface: 1 = smooth; 2 = slightly rough; 3 =moderately rough or ribbed 2 2 Fruit base color (mature-green stage): 1= light green; 2 = light gray-green; 3 3 3 = apple or medium green 4 =yellow green; 5 = dark green Fruit pattern (mature-green stage): 1 =uniform green; 2 = green-shouldered; 1 1 3 = radial stripes on sides offruit Fruit color full ripe: 1 = white; 2 = yellow; 3 = orange; 4 =pink; 5 = red; 5 5 6 = brownish; 7 = greenish; 8 = other Flesh colorfull ripe: 1 = yellow; 2 = pink; 3 = red/crimson; 4 = orange; 5 other 33 Flesh color: 1 = uniform; 2 = with lighter and darker areas in walls 11 locular gel color of table-ripe fruit: 1 = green; 2 = yellow; 3 = red3 3 fruit ripening: 1 = blossom to stem end; 2 = uniform ripening 2 1ripening: 1 = inside out; 2 = uniformity; 3 = outside in 1 1 stem scarsize: 1 = small (Roma); 2 = medium; 3 = large 2 1 core: 1 = coreless(absent or smaller than 6 × 6 mm); 2 = present 2 2 epidermis color: 1 =colorless; 2 = yellow 2 2 epidermis: 1 = normal; 2 = easy-peel 2 2epidermis texture: 1 = tender; 2 = average; 3 = tough 3 3 thickness ofpericarp (cm) 0.9 0.7 Field holding ability no yes Mature fruit firmnessmeasured with dentometer with 5 data point average (P5) 4.47 4.10 Fruitharvestability: 1 = many rotten or broken; 2 = fruit soft, many rottenfruits; 4 5 3 = some rotten fruit; 4 = few rotten fruit; 5 = no rottenfruits, no rejected fruits Disease and pest reaction: 0 = not tested; 1= highly resistant; 2 = resistant, few symptoms; 3 = resistance, fewlesions in number and size; 4 = moderately resistance; 5 = intermediateresistance; 6 = moderate susceptible; 7 = susceptible; 9 = highlysusceptible Virus diseases cucumber mosaic 0 0 curly top 0 0 potato-yvirus 0 0 blotchy ripening 0 0 tobacco mosaic race 0 7 7 tobacco mosaicrace 1 7 7 tobacco mosaic race 2 7 7 tobacco mosaic race 22 7 7 Tomatospotted wilt 2 7 Tomato yellow leaf curl 7 7 Others 0 0 Bacterialdisease Bacterial canker (Corynebacterium michiganense) 0 0 Bacterialsoft rot (Erwinia corotovora) 0 0 Bacterial speck (Pseudomonas tomato)race 0 2 2 Bacterial spot (Xanthomonas vesicatorium) 0 0 Bacterial wilt(Pseudomonas solanacearum) 0 0 Other bacterial disease 0 0 Fungaldiseases Anthracnose (Colletotrichum spp.) 0 0 Brown root rot or corkyroot (Pyrenochaeta lycopersici) 0 0 Collar rot or stem canker(Alternaria solani) 0 0 Early blight defoliation (Alternaria solani) 0 0Fusarium wilt race 1 (F. oxysporum f. lycopersici) 2 2 Fusarium wiltrace 2 (F. oxysporum f. lycopersici) 2 2 Fusarium wilt race 3 (F.oxysporum f. lycopersici) 7 7 Grey leaf spot (Stemphylium spp.) 0 0 Lateblight, race 0 (Phytophthora infestans) 0 0 Late blight, race 1 0 0 Leafmold race 1 (Cladosporiom fulvum) 0 0 Leaf mold race 2 (Cladosporiomfulvum) 0 0 Leaf mold race 3 (Cladosporiom fulvum) 0 0 Leaf mold otherraces 0 0 Nailhead spot (Alternaria tomato) 0 0 Seporia leafspot (S.lycopersici) 0 0 Target leafspot (Corynespora casiicola) 0 0Verticillium wilt race 1 (V. albo-atrum) 2 2 Verticillium wilt race 2 00 Other fungal disease 0 0 Insects and Pests colorado potato beetle (L.decemlineata) 0 0 southern root knot nematode (M. incognia) 2 2 spidermites (Tetranychus spp.) 0 0 sugar beet army worm (s. exigual) 0 0tobacco flea beetle (E. hirtipennis) 0 0 tomato hoernworm (M.quinquemaculata) 0 0 tomato fruitworm (H. zea) 0 0 whitefly (T.vaporariorum) 0 0 Other 0 0 Chemistry and composition of full-ripefruits pH 4.31 4.37 Soluble solids as Brix 5.56 4.83 Seedling (Maturityin number of days) to once harvest 125 days 122 days Fruit season(concentration): 1 = long (Marglobe); 2 = medium (Westover); 3 4 3 =short, concentrated; 4 = very concentrated (UC82) Relative maturity inareas tested: 1 = early; 2 = medium early; 3 = medium; 4, 5 4 4 = mediumlate; 5 = late; 6 = variable Adaptation Culture: 1 = field; 2 =greenhouse 1 1 Principle use(s): 1 = home garden; 2 = fresh market; 3 =whole-pack canning; 3, 4, 5 3, 4, 5 4 = concentrated products; 5 =multiuse; 6 = other Average yield (ton/acre) in California (grower'sfield, 5 data points) 57.26 53.63 Machine harvest: 1 = not adapted; 2 =adapted 2 2 Regions to which adaptation has been demonstrated: 1 =Northeast; 2 = Mid 9 + 11 + 12 9 + 11 + 12 Atlantic; 3 = Southeast; 4Florida; 5 = Great Plains; 6 = south central; 7 = Intermountain West; 8= Northwest; 9 = California (Sacramento and Upper San Joaquin Valley);10 = California (Coastal Areas); 11 = California (Southern San JoaquinValley & desserts); 12 = South American countries

The hybrid tomato plant HMX4909 has shown uniformity and stability forthe traits, within the limits of environmental influence for the traitsas described in the following Variety Descriptive Information. Novariant traits have been observed or are expected for agronomicalimportant traits in tomato hybrid HMX4909.

Example 2 Comparison of New HMX4909 Tomato with Check Variety

In the tables that follow, the traits and characteristics of hybridtomato HMX4909 are given compared to another hybrid. The data collectedare presented for key characteristics and traits. Hybrid tomato HMX4909was tested at numerous locations, with two or more replications perlocation. Information about the hybrid, as compared to several checkhybrid is presented (based primarily on data collected in California,all experiments done under the direct supervision of the applicant).

Table 2 below shows the characteristics of hybrid tomato HMX4909compared to hybrid H8504 as measured in California in August. Column 1identifies the varieties, column 2 described disease resistance package(V is Verticillium resistance, FF is Fusarium Race 2 resistance, N isNematode resistance, P is Pseudomonas Race 0 resistance, Sw is TomatoSpotted Wilt Virus Resistance, Fr is Fusarium Crown rot resistance),column 3 the average of fruit puree acidity measured by pH, column 4 thefruit raw brix or soluble solid content, column 5 the average of JuiceBostwick (JB), column 6 the serum viscosity (Ostwald).

TABLE 2 Variety Disease pH Brix Bostwick Ostwald HMX4909 VFFNSwFr 4.315.56 14.52 571 H8504 VFFNP 4.37 4.83 12.77 868

Table 3 below shows the characteristics of hybrid HMX4909 compared tohybrid H8504 as measured in California in August. Column 1 identifiesthe varieties, column 2 the average single fruit weight (grams), column3 the average fruit firmness as measured by “P5” scale, column 4 theaverage fruit color as measured by ration of a/b ratio of fruit puree,column 5 the average fruit yield of 5 plants (in pounds).

TABLE 3 Variety Frt Wt(g) Firmness (P5) Color YLD AVE HMX4909 97 4.472.15 93 H8504 75 4.10 2.15 85Deposit Information

A deposit of the tomato seed of this invention is maintained byHM.CLAUSE, Inc. Davis Research Station, 9241 Mace Boulevard, Davis,Calif. 95616. In addition, a sample of the hybrid tomato seed of thisinvention has been deposited with the National Collections ofIndustrial, Food and Marine Bacteria (NCIMB), 23 St Machar Drive,Aberdeen, Scotland, AB24 3RY, United Kingdom.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain of the present invention meetsthe criteria set forth in 37 CFR 1.801-1.809, Applicants hereby make thefollowing statements regarding the deposited hybrid tomato HMX4909(deposited as NCIMB Accession No. 42534).

-   1. During the pendency of this application, access to the invention    will be afforded to the Commissioner upon request;-   2. All restrictions on availability to the public will be    irrevocably removed upon granting of the patent under conditions    specified in 37 CFR 1.808;-   3. The deposit will be maintained in a public repository for a    period of 30 years or 5 years after the last request or for the    effective life of the patent, whichever is longer;-   4. A test of the viability of the biological material at the time of    deposit will be conducted by the public depository under 37 CFR    1.807; and-   5. The deposit will be replaced if it should ever become    unavailable.    Access to this deposit will be available during the pendency of this    application to persons determined by the Commissioner of Patents and    Trademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35    U.S.C. § 122. Upon allowance of any claims in this application, all    restrictions on the availability to the public of the variety will    be irrevocably removed by affording access to a deposit of at least    2,500 seeds of the same variety with the NCIMB.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as, an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

What is claimed is:
 1. A seed of hybrid tomato designated HMX4909,wherein a representative sample of seed of said hybrid having beendeposited under NCIMB No.
 42543. 2. A tomato plant, or a part thereof,or a plant cell thereof, produced by growing the seed of claim
 1. 3. Thetomato part of claim 2, wherein the tomato part is selected from thegroup consisting of: a leaf, a flower, a fruit, an ovule, pollen, acell, a rootstock, and a scion.
 4. A tomato plant, or a part, or a plantcell thereof, wherein the tomato plant or a plant regenerated from thepart of the plant cell has all of the physiological and morphologicalcharacteristics of hybrid HMX4909 listed in Table 1 when grown under thesame environmental condition, wherein a representative sample of seed ofsaid hybrid HMX4909 has been deposited under NCIMB No.
 42543. 5. Atomato plant, or a part, or a plant cell thereof, having all of thephysiological and morphological characteristics of hybrid HMX4909,wherein a representative sample of seed of said hybrid having beendeposited under NCIMB No.
 42543. 6. A tissue culture of regenerablecells produced from the plant or plant part of claim 2, wherein a plantregenerated from the tissue culture has all of the physiological andmorphological characteristics of hybrid HMX4909 listed in Table 1 whengrown under the same environmental condition, and wherein arepresentative sample of seed of said hybrid having been deposited underNCIMB No.
 42543. 7. A tomato plant regenerated from the tissue cultureof claim 6, said plant having all the physiological and morphologicalcharacteristics of hybrid HMX4909, wherein a representative sample ofseed of said hybrid having been deposited under NCIMB No.
 42543. 8. Atomato fruit produced from the plant of claim
 2. 9. A method forproducing a tomato fruit comprising a) growing the tomato plant of claim2 to produce a tomato fruit, and b) harvesting said tomato fruit.
 10. Atomato fruit produced by the method of claim
 9. 11. A method forproducing a tomato seed comprising crossing a first parent tomato plantwith a second parent tomato plant and harvesting the resultant tomatoseed, wherein said first parent tomato plant and/or second parent tomatoplant is the tomato plant of claim
 2. 12. A method for producing atomato seed comprising self-pollinating the tomato plant of claim 2 andharvesting the resultant tomato seed.
 13. A method of vegetativelypropagating the tomato plant of claim 2, said method comprising a)collecting part of the plant of claim 2 and b) regenerating a plant fromsaid part.
 14. The method of claim 13 further comprising harvesting afruit from said plant.
 15. A plant obtained from the method of claim 13,wherein the plant has all of the physiological and morphologicalcharacteristics of the plant of claim 2 when grown under the sameenvironmental conditions.
 16. A fruit obtained from the method of claim14.
 17. A method of producing a tomato plant derived from the hybridvariety HMX4909, the method comprising the steps of self-pollinating theplant of claim 2 at least once to produce a progeny plant derived fromthe variety HMX4909.
 18. The method of claim 17 further comprising thestep of: (a) crossing the progeny plant derived from the variety HMX4909with itself or a second tomato plant to produce a seed of progeny plantof a subsequent generation; (b) growing the progeny plant of thesubsequent generation from the seed and crossing the progeny plant ofthe subsequent generation with itself or a second tomato plant and (c)repeating steps (a) or (b) for at least 1 more generation to produce atomato plant further derived from the tomato hybrid variety HMX4909. 19.A method of producing a tomato plant derived from the hybrid varietyHMX4909, the method comprising the steps of crossing the plant of claim2 with a second tomato plant to produce a progeny plant derived from thevariety HMX4909.
 20. The method of claim 19 further comprising the stepof: (a) crossing the progeny plant derived from the variety HMX4909 withitself or a second tomato plant to produce a seed of progeny plant of asubsequent generation; (b) growing the progeny plant of the subsequentgeneration from the seed and crossing the progeny plant of thesubsequent generation with itself or a second tomato plant and (c)repeating step (a) or (c) for at least 1 more generation to produce atomato plant further derived from the tomato hybrid variety HMX4909. 21.A method for producing a transgenic tomato plant, the method comprisingcrossing the tomato plant of claim 2 with a second tomato plantcontaining a transgene, wherein the transgene of said second tomatoplant is integrated into the genome of the tomato plant progenyresulting from said cross.
 22. A tomato plant comprising a transgene andotherwise all of the physiological and morphological characteristics ofhybrid tomato plant HMX4909 listed in Table 1 when grown under the sameenvironmental conditions, wherein a representative sample of seed ofHMX4909 has been deposited under NCIMB
 42543. 23. The plant of claim 22,wherein the transgene confers said plant with a trait selected from thegroup consisting of male sterility, male fertility, herbicideresistance, insect resistance, disease resistance, increased sweetness,increased sugar content, increased flavor, improved ripening control,and improved salt tolerance compared to hybrid tomato plant HMX4909 whengrown under the same environmental conditions.